CN117441103A

CN117441103A - Novel medical technology using follicular T cells

Info

Publication number: CN117441103A
Application number: CN202280022067.8A
Authority: CN
Inventors: 山崎晶; 陆修远; 细野裕贵; 石塚茂宜; 山下和男; 尼可拉·克劳德·保罗·萨克斯
Original assignee: Hongtai Biotechnology Co ltd; Osaka University NUC
Current assignee: Hongtai Biotechnology Co ltd; Osaka University NUC
Priority date: 2021-03-16
Filing date: 2022-03-15
Publication date: 2024-01-23
Also published as: EP4310171A1; WO2022196701A1; JPWO2022196701A1

Abstract

The present invention provides a novel medical technique using follicular T cells. The present invention provides a method for producing follicular T cells specific for a disease, based on the finding of a common TfhTCR (Public TfhTCR) specific for a common disease factor of each patient, the method comprising a step of determining a disease-related factor or a part thereof having the ability to trigger follicular T cells specific for a disease-related factor, a step of using the disease-related factor or the part thereof to trigger follicular T cells specific for a disease-related factor, and a step of obtaining follicular T cells specific for the disease.

Description

Novel medical technology using follicular T cells

Technical Field

The present invention relates to a novel medical technology using follicular T cells. More specifically, the present invention relates to a technique for diagnosing a disease using follicular T cells as an index.

Background

The innate immune response is generated against various disease factors, but there are many unknown points in this. Thus, understanding the acquired immune response to disease factors becomes critical in predicting long-term immunity and providing information for vaccine design.

Disclosure of Invention

[ problem ] to be solved by the invention

The present inventors have found that when there is a certain infective element (virus, etc.), there is a specific public TfhTCR (Public TfhTCR) among co-infective elements of various patients. The inventors found that by recombining TCR alpha and beta chains, it was confirmed that the antigen presented by the frequently found allele was a T cell epitope. The inventors have found that the use of this TCR clonotype can be used for diagnosis.

Accordingly, the present invention provides the following.

(item 1)

A method for evaluating a disease in a subject, the method comprising the step of determining the level or amount of follicular T cells reactive with a disease-related factor of the disease in the subject.

(item 2)

The method of any one of the above items, further comprising performing at least 1 step selected from the group consisting of: the history of the disease, the effectiveness of a prophylactic or vaccine against the disease, the effectiveness of a therapeutic against the disease, the ability to defend against a secondary disease, the disease condition and the risk of developing the disease.

(item 3)

The method of any one of the above items, further comprising the step of examining or diagnosing the disease of the subject.

(item 4)

The method according to any one of the above items, further comprising the step of evaluating infection history of the disease of the subject.

(item 5)

The method according to any one of the above items, further comprising the step of evaluating vaccine effectiveness and reinfection defense ability of the disease in the subject.

(item 6)

The method according to any one of the preceding items, further comprising the step of performing an evaluation of the effectiveness of a cancer immune drug of the disease of the subject.

(item 7)

The method according to any one of the above items, wherein the disease-related factor is an autoimmune disease-related factor, and the measurement further comprises a step of measuring the level or amount of follicular T cells reactive with the autoimmune disease-related factor, and a step of evaluating the risk and disease condition of the autoimmune disease associated with the autoantibody in the subject.

(item 8)

The method according to any of the preceding items, wherein the assay is performed according to enzyme-linked immunosorbent assay, i.e. ELISPOT (Enzyme Linked Immunospot Assay), flow cytometry, re-stimulation of Peripheral Blood Mononuclear Cells (PBMCs), new generation sequencer/genetic examination (histology), immunostaining (pathology), in situ analysis.

(item 9)

The method of any of the above items, the method further comprising: comparing the measured level, spatial distribution or amount of follicular T cells reactive with the disease-related factor with the average or median level of the level, spatial distribution or amount of follicular T cells reactive with the disease-related factor of a subject not suffering from the disease.

(item 10)

The method of any one of the above items, wherein the disease is an infection or cancer.

(item 11)

The method of any one of the above items, wherein the disease is a viral infection.

(item 12)

The method of any one of the above items, wherein the disease is an allergy or an autoimmune disease.

(item 13)

A reagent or device for evaluating a disease in a subject, the reagent or device comprising a reagent or device for determining the level, spatial distribution or amount of follicular T cells reactive with a disease-related factor of the disease in the subject.

(item 14)

The reagent or device of any one of the above items comprising a reagent that specifically reacts with follicular T cells.

(item 15)

The reagent or device of any one of the above items, which is a device for performing ELISPOT, flow cytometry, re-stimulation of Peripheral Blood Mononuclear Cells (PBMCs), new generation sequencer/genetic testing (histology), immunostaining (pathology), in situ analysis.

(item 16)

The agent or device of any of the above items, wherein the disease is an infection or cancer.

(item 17)

The agent or device of any one of the above items, wherein the disease is a viral infection.

(item 18)

The agent or device of any of the above items, wherein the disease is an allergy or autoimmune disease.

(item 19)

A system for evaluating a disease of a subject, comprising a reagent or device for measuring the level, spatial distribution or amount of follicular T cells reactive with a factor associated with the disease of the subject, and an evaluation unit for evaluating the disease based on the measurement result of the reagent or device.

(item 20)

The system according to any one of the preceding items, wherein the evaluation section is configured to perform at least 1 selected from the group consisting of: the evaluation of the history of the disease, the evaluation of the effectiveness of a prophylactic or vaccine for the disease, the evaluation of the effectiveness of a therapeutic for the disease, the evaluation of the ability to defend against the disease again, the evaluation of the condition of the disease, and the evaluation of the risk of developing the disease.

(item 21)

The system according to any one of the above items, further comprising an examination/diagnosis unit that examines or diagnoses the disease of the subject.

(item 22)

The system according to any one of the above items, further comprising an infection history evaluation unit that evaluates infection history of the disease of the subject.

(item 23)

The system according to any one of the above items, further comprising a vaccine effectiveness evaluation and reinfection defense capability evaluation unit for evaluating vaccine effectiveness and reinfection defense capability of the disease of the subject.

(item 24)

The system according to any one of the above items, further comprising a cancer immune drug effectiveness evaluation unit that evaluates the effectiveness of the cancer immune drug of the disease of the subject.

(item 25)

The system according to any one of the above items, wherein the disease-related factor is an autoimmune disease-related factor, and the system comprises a follicular T cell measurement unit for measuring the level, spatial distribution, or amount of follicular T cells reactive with the autoimmune disease-related factor, and an autoimmune disease risk evaluation unit for performing risk evaluation, disease condition evaluation, and autoimmune disease condition evaluation of the autoimmune disease associated with the autoantibody in the subject.

(item 26)

The system of any one of the above items, wherein the reagent or device comprises a reagent that specifically reacts with follicular T cells.

(item 27)

The system according to any of the preceding items, wherein the device is a device for performing ELISPOT, flow cytometry, re-stimulation experiments of Peripheral Blood Mononuclear Cells (PBMCs), new generation sequencer/genetic examination (histology), immunostaining (pathology), in situ analysis.

(item 28)

The system of any of the above items, the system further comprising: and a comparison unit for comparing the measured level, spatial distribution or amount of follicular T cells reactive with the disease-related factor with the average value or median of the levels, spatial distribution or amount of follicular T cells reactive with the disease-related factor of the subject not suffering from the disease.

(item 29)

The system of any of the above items, wherein the disease is an infection or cancer.

(item 30)

The system of any one of the above items, wherein the disease is a viral infection.

(item 31)

The system of any one of the above items, wherein the disease is an allergy or an autoimmune disease.

(item 32)

A method for evaluating a disease in a subject, the method comprising the steps of providing information about the level, spatial distribution or amount of follicular T cells reactive with a disease-related factor of the disease in the subject, and evaluating the information about the level, spatial distribution or amount of follicular T cells for the disease.

(item 33)

The method according to any one of the preceding claims, wherein the step of evaluating further comprises the step of performing at least 1 selected from the group consisting of: the evaluation of the history of the disease, the evaluation of the effectiveness of a prophylactic or vaccine for the disease, the evaluation of the effectiveness of a therapeutic for the disease, the evaluation of the ability to defend against the disease again, the evaluation of the condition of the disease, and the evaluation of the risk of developing the disease.

(item 34)

The method according to any one of the above items, comprising a step of evaluating infection history of the disease of the subject.

(item 35)

The method according to any one of the above items, which comprises a step of evaluating vaccine effectiveness, reinfection defense ability of the disease of the subject.

(item 36)

The method according to any one of the preceding items, comprising the step of evaluating the effectiveness of a cancer immune drug of the disease of the subject.

(item 37)

The method according to any one of the above items, wherein the disease-related factor is an autoimmune disease-related factor, and the measurement comprises a step of measuring the level or amount of follicular T cells reactive with the autoimmune disease-related factor, and a step of evaluating the risk and disease condition of the autoimmune disease associated with the autoantibody in the subject.

(item 38)

The method of any one of the above items, wherein the step of providing information is performed according to ELISPOT, flow cytometry, re-stimulation assays of Peripheral Blood Mononuclear Cells (PBMCs), new generation sequencer/genetic testing (histology), immunostaining (pathology), in situ analysis.

(item 39)

The method of any of the above items, the method further comprising: and comparing the information on the level, spatial distribution or amount of the follicular T cells reactive with the disease-related factor of the disease of the subject with the average value or median of the information on the level, spatial distribution or amount of the follicular T cells reactive with the disease-related factor of the disease of the subject not suffering from the disease.

(item 40)

(item 41)

(item 42)

(item 43)

A program for installing a method for evaluating a disease of a subject in a computer, the program comprising a computer-readable code, the program comprising a step of providing information on the level, spatial distribution or amount of follicular T cells reactive with a disease-related factor of the disease of the subject, and a step of evaluating the information on the level, spatial distribution or amount of follicular T cells for the disease.

(item 44)

The program according to any one of the above items, wherein the step of evaluating further comprises a step of performing at least 1 selected from the group consisting of: the evaluation of the history of the disease, the evaluation of the effectiveness of a prophylactic or vaccine for the disease, the evaluation of the effectiveness of a therapeutic for the disease, the evaluation of the ability to defend against the disease again, the evaluation of the condition of the disease, and the evaluation of the risk of developing the disease.

(item 45)

The program according to any one of the above items, wherein the method comprises a step of evaluating infection history of the disease of the subject.

(item 46)

The program according to any one of the above items, wherein the method comprises a step of evaluating vaccine effectiveness and reinfection defense ability of the disease of the subject.

(item 47)

The program according to any one of the above items, wherein the method comprises a step of evaluating the effectiveness of the cancer immune drug of the disease of the subject.

(item 48)

The program according to any one of the preceding items, wherein the disease-related factor is an autoimmune disease-related factor, the method comprising: the measurement is a step of measuring the level or amount of follicular T cells reactive with the autoimmune disease-related factor, and a step of evaluating the risk and disease condition of the autoimmune disease accompanied by the autoantibody in the subject.

(item 49)

The program according to any one of the above items, wherein the step of providing information is performed according to ELISPOT, flow cytometry, re-stimulation of Peripheral Blood Mononuclear Cells (PBMC), new generation sequencer/genetic testing (histology), immunostaining (pathology), in situ analysis.

(item 50)

The program of any one of the preceding items, wherein the method further comprises: and comparing the information on the level, spatial distribution or amount of the follicular T cells reactive with the disease-related factor of the disease of the subject with the average value or median of the information on the level, spatial distribution or amount of the follicular T cells reactive with the disease-related factor of the disease of the subject not suffering from the disease.

(item 51)

The procedure of any one of the above items, wherein the disease is an infection or cancer.

(item 52)

The program of any one of the above items, wherein the disease is a viral infection.

(item 53)

The procedure according to any one of the preceding items, wherein the disease is an allergy or an autoimmune disease.

(item 54)

A computer-readable recording medium in which the program of any one of the above items is stored.

(item 55)

The present invention also provides the following items.

(item A1)

A pharmaceutical composition for use in the treatment or prophylaxis of a disease, said composition having the ability to elicit follicular T cells reactive with a disease related factor of the disease and comprising the disease related factor or a portion or functional equivalent thereof.

(item A2)

A vaccine against the above-mentioned disease, said vaccine having the ability to elicit follicular T cells reactive with a factor associated with said disease and comprising the factor associated with the disease or a portion or functional equivalent thereof.

(item A3)

The composition or vaccine of any one of the above items, which preferentially induces follicular T cells.

(item A4)

A pharmaceutical composition for use in the treatment or prophylaxis of the above-mentioned diseases, said composition comprising follicular T cells which are reactive with a factor associated with said disease.

(item A5)

A cellular vaccine against the above-mentioned disease, said cellular vaccine comprising follicular T cells reactive with a factor associated with said disease.

(item A6)

The composition or vaccine of any of the above items, wherein follicular T cells reactive with the disease-related factor are reactive at least with the disease-related factor.

(item A7)

The composition or vaccine of any of the above items, wherein the disease is an infection or cancer.

(item A8)

The composition or vaccine of any of the above items, wherein the disease is a viral infection.

(item A9)

The composition of any one of the above items, which induces HLA-type specific follicular T cells.

(item A10)

The composition of any of the above items, wherein the disease-related factor or a portion or functional equivalent thereof comprises at most 20 amino acids.

(item A11)

The composition of any of the above items, wherein the follicular T cells are public follicular T cells.

(item A12)

A method of producing follicular T cells reactive with said disease, said method comprising the step of determining said disease-associated factor or a portion or functional equivalent thereof having the ability to elicit follicular T cells reactive with said disease-associated factor,

A step of causing specific follicular T cells for the disease using the disease-related factor or a part or functional equivalent thereof,

and a step of obtaining follicular T cells that are reactive with the disease.

(item 13)

A method of screening for follicular T cells reactive with said disease, the method comprising:

a) A step of providing a follicular T cell mass,

B) A step of bringing the follicular T cell mass into contact with the disease-related factor,

C) Measuring the reactivity of the follicular T cells to the disease-related factor, and a method for measuring the reactivity of the follicular T cells to the disease-related factor

D) Selecting follicular T cells exhibiting reactivity of a predetermined value or more with respect to the disease-related factor from the follicular T cell population.

(item A14)

The method according to the above item, wherein the step of C) measuring is performed by ELISPOT, flow cytometry, and a Peripheral Blood Mononuclear Cell (PBMC) restimulation assay.

(item B1)

A polypeptide for presenting a factor associated with said disease or a portion or functional equivalent thereof, said polypeptide having the ability to elicit follicular T cells reactive with said factor associated with said disease.

(item B2)

The polypeptide of any one of the above items, wherein the polypeptide preferentially induces follicular T cells.

(item B3)

The polypeptide of any one of the above items, wherein follicular T cells reactive with the disease-related factor are reactive with at least the disease-related factor.

(item B4)

The polypeptide of any one of the above items, wherein the disease is an infection or cancer.

(item B5)

The polypeptide of any one of the above items, wherein the disease is a viral infection.

(item B6)

The polypeptide of any one of the above items, wherein the polypeptide induces HLA-type specific follicular T cells.

(item B7)

The polypeptide of any one of the above items, wherein the disease-related factor or a portion or functional equivalent thereof comprises at most 20 amino acids.

(item B8)

The polypeptide of any one of the above items, wherein the follicular T cell is a public follicular T cell.

(item C1)

An antibody that specifically binds to a TCR that interacts with a factor associated with the disease or a portion or functional equivalent thereof, the antibody having the ability to elicit follicular T cells reactive with the factor associated with the disease.

(item C2)

The antibody of any one of the above items, wherein the antibody preferentially induces follicular T cells.

(item C3)

The antibody of any one of the above items, wherein follicular T cells reactive with the disease-related factor are reactive at least with the disease-related factor.

(item C4)

The antibody of any one of the above items, wherein the disease is an infection or cancer.

(item C5)

The antibody of any one of the above items, wherein the disease is a viral infection.

(item C6)

The antibody of any one of the above items, wherein the antibody induces HLA-type specific follicular T cells.

(item C7)

An antibody according to any one of the preceding items, wherein the disease-related factor or a portion or functional equivalent thereof comprises a maximum of 20 amino acids.

(item C8)

The antibody of any one of the above items, wherein the follicular T cell is a public follicular T cell.

(item D1)

A pharmaceutical composition for diagnosing a disease, said pharmaceutical composition having the ability to elicit follicular T cells reactive with a factor associated with said disease, wherein the factor associated with the disease or a portion or functional equivalent thereof is used as an indicator.

(item D2)

The pharmaceutical composition of any one of the above items, wherein the pharmaceutical composition preferentially induces follicular T cells.

(item D3)

The pharmaceutical composition of any one of the above items, wherein follicular T cells reactive with the disease-related factor are reactive with at least the disease-related factor.

(item D4)

The pharmaceutical composition according to any of the above items, wherein the disease is an infection or cancer.

(item D5)

The pharmaceutical composition according to any of the above items, wherein the disease is a viral infection.

(item D6)

The pharmaceutical composition of any one of the above items, wherein the pharmaceutical composition induces HLA-type specific follicular T cells.

(item D7)

The pharmaceutical composition according to any of the preceding items, wherein the disease-related factor or a part or functional equivalent thereof comprises at most 20 amino acids.

(item D8)

The pharmaceutical composition of any of the above items, wherein the follicular T cells are public follicular T cells.

(item E1)

A composition for enhancing the acquisition of immunity to said disease, said composition comprising follicular T cells reactive with a factor associated with said disease.

(item E2)

The composition or vaccine of any of the above items, wherein follicular T cells reactive with the disease-related factor are reactive with at least the disease-related factor.

(item E3)

(item E4)

(item E5)

The composition or vaccine of any of the above items, wherein the follicular T cells are public follicular T cells.

(item X1)

A method for testing a subject for a disease-related item, the method comprising:

a step of measuring the level or amount of follicular T cells reactive with a factor associated with the disease of the subject, and

and comparing the level or amount with a predetermined reference.

(item X2)

The method of any of the above items, wherein the benchmark is a benchmark related to at least 1 selected from the group consisting of: the history of the disease, the effectiveness of a prophylactic or vaccine for the disease, the effectiveness of a therapeutic for the disease, the ability to defend against the disease again, the condition of the disease, and the risk of developing the disease.

(item X3)

The method of any of the above items, wherein the benchmark is a benchmark for the disease of the subject.

(item X4)

The method according to any one of the above items, wherein the criterion is a criterion for infection history of the disease of the subject.

(item X5)

The method according to any one of the above items, wherein the criterion is a criterion concerning the effectiveness of a vaccine against the disease of the subject, reinfection defense ability.

(item X6)

The method of any one of the above items, wherein the benchmark is a benchmark for the effectiveness of a cancer immune drug against the disease of the subject.

(item X7)

The method according to any one of the above items, wherein the disease-related factor is an autoimmune disease-related factor, and the measurement comprises a step of measuring the level or amount of follicular T cells reactive with the autoimmune disease-related factor, and the criterion is a criterion for the risk of, or the disease condition of, the autoimmune disease associated with the autoantibody of the subject.

(item X8)

The method of any one of the above items, wherein the determining is performed according to ELISPOT, flow cytometry, re-stimulation of Peripheral Blood Mononuclear Cells (PBMCs), new generation sequencer/genetic testing (histology), immunostaining (pathology), in situ analysis.

(item X9)

The method of any one of the preceding claims, wherein the comparing further comprises the step of comparing the determined level, spatial distribution or amount of follicular T cells reactive with the disease-related factor with the average or median level of follicular T cells reactive with the disease-related factor of a subject not suffering from the disease.

(item X10)

(item X11)

(item X12)

(item X13)

(item X14)

(item X15)

The reagent or device of any one of the above items, wherein the device is a device for performing ELISPOT, flow cytometry, re-stimulation of Peripheral Blood Mononuclear Cells (PBMCs), new generation sequencer/genetic testing (histology), immunostaining (pathology), in situ analysis.

(item X16)

(item X17)

(item X18)

(item X19)

A system for testing a subject for a disease-related item, the system comprising: a reagent or device for measuring the level, spatial distribution or amount of follicular T cells reactive with a disease-related factor of the disease in the subject, and a comparing unit for comparing the measurement result obtained by the reagent or device with a predetermined standard.

(item X20)

The system of any of the above items, wherein the benchmark is a benchmark with respect to at least 1 selected from the group consisting of: the history of the disease, the effectiveness of a prophylactic or vaccine against the disease, the effectiveness of a therapeutic against the disease, the ability to defend against a secondary disease, the condition of the disease, and the risk of developing the disease.

(item X21)

The system of any of the above items, wherein the benchmark is a benchmark for the disease of the subject.

(item X22)

The system according to any one of the preceding claims, wherein the criterion is a criterion for the disease history of the subject.

(item X23)

The system according to any one of the above items, wherein the criterion is a criterion concerning the effectiveness of a prophylactic agent or vaccine against the disease of the subject and reinfection preventing ability.

(item X24)

The system of any of the above items, wherein the benchmark is a benchmark for the effectiveness of a cancer immune drug against the disease of the subject.

(item X25)

The system according to any one of the above items, wherein the disease-related factor is an autoimmune disease-related factor, and the system further comprises a follicular T cell measurement unit for measuring a level, spatial distribution, or amount of follicular T cells reactive with the autoimmune disease-related factor, and the criterion is a criterion for a risk or a disease condition of the subject associated with an autoimmune disease by the autoantibody.

(item X26)

The system of any one of the above items, wherein the reagent or device comprises a reagent that specifically reacts to follicular T cells.

(item X27)

(item X28)

The system according to any one of the above items, wherein the comparing unit compares the measured level, spatial distribution or amount of follicular T cells reactive with the disease-related factor with the average value or median of the levels, spatial distributions or amounts of follicular T cells reactive with the disease-related factor of the subject not suffering from the disease.

(item X29)

(item X30)

(item X31)

(item X32)

a step of providing information on the level, spatial distribution or amount of follicular T cells reactive with a disease-related factor of the disease in the subject, and

and comparing the information on the level, spatial distribution or amount of the follicular T cells with a predetermined standard.

(item X33)

The method of any of the above items, wherein the benchmark is a benchmark related to at least 1 selected from the group consisting of: the history of the disease, the effectiveness of a prophylactic or vaccine against the disease, the effectiveness of a therapeutic against the disease, the ability to defend against a secondary disease, the assessment of the condition of the disease, and the risk of developing the disease.

(item X34)

(item X35)

(item X36)

(item X37)

(item X38)

The method of any one of the above items, wherein the step of providing information is performed by ELISPOT, flow cytometry, re-stimulation of Peripheral Blood Mononuclear Cells (PBMCs), new generation sequencer/genetic testing (histology), immunostaining (pathology), in situ analysis.

(item X39)

The method according to any one of the preceding claims, wherein the comparing further comprises the step of comparing the information about the level, spatial distribution or amount of follicular T cells reactive with the disease-related factor of the disease of the subject with the average or median of the information about the level, spatial distribution or amount of follicular T cells reactive with the disease-related factor of the disease of the subject not suffering from the disease.

(item X40)

(item X41)

(item X42)

(item X43)

A program for installing a method for testing matters related to a disease of a subject in a computer, the program comprising a computer-readable code, the program comprising a step of providing information on the level, spatial distribution or amount of follicular T cells reactive with a disease-related factor of the disease of the subject, and a step of comparing the information on the level, spatial distribution or amount of follicular T cells with a predetermined standard.

(item X44)

The program according to any one of the preceding items, wherein the criterion is a criterion related to at least 1 selected from the group consisting of: the history of the disease, the effectiveness of a prophylactic or vaccine against the disease, the effectiveness of a therapeutic against the disease, the ability to defend against a secondary disease, the assessment of the condition of the disease, and the risk of developing the disease.

(item X45)

The program according to any one of the above items, wherein the criterion is a criterion for infection history of the disease of the subject.

(item X46)

The program according to any one of the above items, wherein the criterion is a criterion for vaccine effectiveness, reinfection defense ability of the disease of the subject.

(item X47)

The program of any one of the above items, wherein the benchmark is a benchmark for the effectiveness of the cancer immune drug of the disease of the subject.

(item X48)

The program according to any one of the preceding items, wherein the disease-related factor is an autoimmune disease-related factor, the method comprising: the measurement is a step of measuring the level or amount of follicular T cells that are reactive with the autoimmune disease-related factor, and the criterion is a criterion for the risk or condition of autoimmune disease associated with the autoantibodies in the subject.

(item X49)

The program according to any one of the above items, wherein the step of providing information is performed by ELISPOT, flow cytometry, re-stimulation of Peripheral Blood Mononuclear Cells (PBMC), new generation sequencer/genetic testing (histology), immunostaining (pathology), in situ analysis.

(item X50)

The program of any of the above claims, wherein the comparing further comprises: and comparing the information on the level, spatial distribution or amount of the follicular T cells reactive with the disease-related factor of the disease of the subject with the average value or median of the information on the level, spatial distribution or amount of the follicular T cells reactive with the disease-related factor of the disease of the subject not suffering from the disease.

(item X51)

(item X52)

(item X53)

(item X54)

In the present invention, one or more of the features described above are intended to be provided in further combination in addition to the combination already explicitly described. Further embodiments and advantages of the present invention will be appreciated to those skilled in the art upon reading and understand the following detailed description, if necessary.

[ Effect of the invention ]

The present invention provides a follicular helper T cell (Tfh) specific for a composition comprising an epitope specific for a disease factor for inducing follicular T cells based on the discovery of a common specificity among common disease factors in various patients TfhTCR (Public TfhTCR).

Drawings

Fig. 1: FIG. 1 is a graph showing the TCR expressed in Tfh cells from a patient with COVID-19 as determined. UMAP projection of 18661T cells from PBMC of patient. Each dot corresponds to a single cell (single cell). Tfh clusters shown in the UMAP plot were tagged using standard CD200, ICOS, CD40LG, PDCD1, CXCL13 and CXCR5 as Tfh cell markers.

Fig. 2: FIG. 2 is a diagram showing the arrangement of TCR CDR3 sequences of clone 1 and clone 2.

Fig. 3-1: FIG. 3 is a graph showing that TCRs cloned from Tfh cells of a patient are reactive with peptides derived from SARS-CoV-2S protein. T cell hybridomas expressing TCR of clonotype 1/2 (clone 1 and clone 2, respectively) were stimulated with inactivated virus (corresponding to 1. Mu.g/ml S protein), recombinant S protein (1. Mu.g/ml), S peptide pool and M+N peptide pool (1. Mu.g/ml each peptide) (blank histogram, respectively) in the presence (A) or absence (B) of APCs from the corresponding donors for 20 hours, in a non-stimulated state (painted histogram), and analyzed for CD69 expression (FIGS. 3A and B).

Fig. 3-2: FIG. 3 is a graph showing that TCRs cloned from Tfh cells of a patient are reactive with peptides derived from SARS-CoV-2S protein. Clone 1 and clone 2 were stimulated with pools #1 and #2 (1 μg/ml of each peptide) in the presence of APC of the same origin over a period of 20 hours (fig. 3C). Clone 1 and clone 2 were stimulated with the S peptide pool #2 and the S peptide pool PepTivator (registered trademark), both 0.3 μg/ml per peptide, over a period of 20 hours in the presence of APCs of the same origin, followed by staining for CD69 (FIG. 3D). Clone 1 and clone 2 were stimulated with S peptide pool #2 (1 μg/ml per peptide) in the presence of APCs from different donors over a period of 20 hours, and then analyzed for CD69 expression (FIG. 3E).

Fig. 4: FIG. 4 is a graph showing the relative positions of coverage of peptide pools.

Fig. 5-1: FIG. 5 is a graph showing the determined HLA and S protein peptides recognized by S protein responsive TCRs. T cell hybridomas expressing TCR of clonotype 1/2 (clone 1 and clone 2, respectively) were stimulated with S peptide pool #2 (0.3 μg/ml of each peptide) in the presence of APCs from other donor PBMCs with the same DP or DR/DQ alleles of the original individual (FIG. 5A). Clones 1 and 2 (FIG. 5B) were stimulated with S peptide pool #2 (0.3 μg/ml per peptide) in the presence of HEK 293T cells expressing the indicated HLA.

Fig. 5-2: FIG. 5 is a graph showing the determined HLA and S protein peptides recognized by S protein responsive TCRs. Predicted by NetMHC, having the data predicted by DRB1 x 15:01 to a portion of the S protein containing the epitopes of clones 1 and 2 (red: strongly binding peptide, grey: weakly binding peptide) (FIG. 5C). Sequences of two strong binding peptides were synthesized (fig. 5D). Clones 1 and 2 were stimulated in the absence of stimulation (painted histogram) or with S peptide pool #2 (0.3 μg/ml peptide) and mono peptide (1 μg/ml) (blank histogram) over 20 hours in the presence of APC of different origin, and then CD69 was stained (fig. 5E).

Fig. 6-1: in FIG. 6, S _864-882 Is a SARS-CoV-2 specific peptide widely recognized by healthy people and convalescent patients with COVID-19. T cell hybridomas expressing TCR of clonotype 1/2 (clone 1 and clone 2, respectively) were co-cultured with pools of peptides from the APC and HCoV-OC 43S proteins (0.3 μg/ml per peptide) (FIG. 6A). S is S _864-882 The sequence arrangement of SARS-CoV-2 in the corresponding region of human coronavirus (FIG. 6B). Peptides S predicted by NetMHC 4.0 server _864-882 DRB1 x 15:01 and 15:02 (red: strong binding peptide, gray: weak binding peptide) (fig. 6C).

Fig. 6-2: in FIG. 6, S _864-882 Is a SARS-CoV-2 specific peptide widely recognized by healthy people and convalescent patients with COVID-19. With DRB1 x 15:02, clones 1 and 2 were stimulated with a single peptide and analyzed for CD69 expression (fig. 6D). Distribution of clonotype 1/2 in public databases of healthy donors and convalescent-19 patients. The upper panel is the percentage of individuals with clonotype 1/2 in both populations and the lower panel is the increase in clonotype 1/2 in each individual of both populations (fig. 6E).

Fig. 7: FIG. 7 shows the immortalization of a mixture of B cells from a patient by CD4 positive cells and EBV infection by SARS-CoV-2S _864-882 The concentration of IL-21 in the medium was higher when stimulated with the peptide than when not stimulated.

Fig. 8: fig. 8 is a diagram showing an example of the configuration of the system 1000.

Fig. 9: fig. 9 is a diagram showing an example of the configuration of the user device 100.

Fig. 10: fig. 10 shows an example of the configuration of the user device 200.

Detailed Description

The following description is presented while showing the best mode of the invention. Throughout this specification, unless otherwise indicated, the singular forms "a", "an" and "the" are to be construed to include plural forms of the concepts as well. Thus, where not otherwise mentioned, the singular forms of articles (e.g., "a," "an," "the," etc. in the case of English) are to be construed to also include the plural forms of the concepts thereof. Furthermore, the words used in the specification should be understood as being used in a sense commonly used in the art unless otherwise noted. Accordingly, unless defined otherwise, all terms of art and science and technology used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In case of conflict, the present specification, including definitions, will control.

The definitions and/or basic technical content of the terms particularly used in the present specification are appropriately described below.

(definition etc.)

"follicular T cells" in this specification refer to: helper T cells possessing a common TCR that inhibit maturation and activation of B cells, and antibody production. Also referred to as follicular helper T cells, follicular T cells and follicular helper T cells are used interchangeably herein.

In the present specification, "specificity" means: the antibody or a fragment thereof or a cell containing the same has a property of recognizing and binding to a target with higher affinity than any other target.

In the present specification, "reactivity" (follicular T cells) to a subject means: the follicular T cells that react with at least the subject and that are activated include substances that can react with substances other than the subject (cross-reactive substances), substances that have low reactivity with substances other than the subject (substances that are preferential to the subject), or substances that have no reactivity (substances that are specific to the subject), regardless of whether they react with substances other than the subject.

In the present specification, "preferential induction" means: the subject is induced at a relatively high rate, for example, at 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 100% or more.

In the present specification, "disease factor" means: in addition to sources of infection such as viruses, there are factors causing immune abnormality in the case of neoplasms such as cancer and autoimmune diseases.

In the present specification, "epitope" means: molecules or moieties known as antigenic determinants that are recognized by the immune system such as antibodies, B cells or T cells may also refer to sites in the antigenic molecule to which the antibody or lymphocyte receptor binds. As used herein, an "epitope" is a molecule that can bind to a binding moiety (e.g., an antibody or antigen binding fragment thereof) described in the specification. In general, epitopes are composed of chemically active surface groups of molecules such as amino acids or sugar side chains, often with specific three-dimensional structural features, and possess specific charge characteristics. In the present specification, the method for determining an epitope is a method known in the art, and when a primary structure of a nucleic acid or an amino acid is provided, a person skilled in the art can determine such an epitope by such a known general technique. "antigen" in the present specification means: any matrix capable of specifically binding to an antibody molecule. "immunogen" (immunogen) in this specification means: an antigen capable of eliciting lymphocyte activation that produces an antigen-specific immune response.

In the present specification, the term "probe presenting an epitope to HLA" means: by using a probe for presenting an epitope to HLA and measuring the presence or absence of a cell bound to the probe, the presence or absence of an immune system can be checked. Although the probe is used, the HLA and the epitope peptide may be integrated as a polypeptide, but the peptide may be attached to the HLA instead of being integrated.

In this specification, "specifically binds to TCR" means: specifically binds to T Cell Receptors (TCRs).

In the present specification, "booster acquisition" means: promoting natural immunity against antigen and obtaining immunity. Examples thereof include promotion of maturation and activation of B cells and control of antibody production by follicular T cells.

In the present specification, "defensive antibody" means: the antibodies that can be observed in active or passive immunization bear the immunity to infectious pathogens, in particular the ability to prevent viral infection.

In the present specification, "common follicular T cells" means: helper T cells with TCRs shared between different humans that control maturation and activation of B cells, antibody production. Can be used interchangeably with public follicular helper T cells and public Tfh cells.

In this specification, "operatively connected" means: the polypeptide encoded by the nucleic acid is linked to an element such as a promoter under the control of the element so as to be expressed in a state that shows the biological activity of the polypeptide.

In the present specification, the term "disease" is used interchangeably with "disorder" and "symptom" in the present invention, and refers to a state of physical and mental disorder or discomfort of a human or an animal in a broader sense, and any state of disease, disorder, various symptoms, and the like, which are not specifically defined and cannot be said to be healthy.

In the present specification, "evaluation" of a disease or the like means: confirming the quality of things, properties, capabilities, etc. for their subject disease from some sides, etc. determines their value or meaning, etc., including, for example, but not limited to: evaluation of disease history, evaluation of the effectiveness of a prophylactic agent or vaccine for a disease, evaluation of the effectiveness of a therapeutic agent for a disease (for example, a cancer immune drug such as an immune checkpoint inhibitor in the case of cancer), evaluation of the ability to defend against a disease again (for example, reinfection in the case of infection), evaluation of the condition of a disease (for example, cancer, infection, autoimmune disease or allergy), and evaluation of the risk of developing the above-mentioned disease.

In the present specification, "disease-related factor" means: in addition to factors that directly cause a disease or factors that cause an indirect cause such as the above-mentioned direct factors, as well as resulting factors that produce results of a disease, factors that are produced simultaneously, factors that are produced in association, and the like, any factor that changes in the disease state of a subject as compared with a factor that causes a disease in a non-disease state. The factor causing the disease is referred to as "disease cause factor", and in this case, for example, in the case of an infectious disease, it means a virus, a bacterium, a protozoa, a mycoplasma, etc. which are causative factors or infectious factors, in the case of an autoimmune disease such as allergy, it means a substance (causative substance) which becomes an immunogen such as an allergen, and in the case of a neoplasm, it includes tumor cells, toxic substances, etc. On the other hand, the factors that produce disease results are also referred to as "disease result factors", and in this case, include factors, metabolites, and the like that are related to the immune response of the subject.

"reactivity" in "follicular T cells reactive with disease-related factors" in the present specification means: when follicular T cells are subjected to conditions that interact with a disease-related factor, other factors change in the presence of that factor as compared to when that factor is absent. Follicular T cells reactive with disease-related factors refer to: the follicular T cells undergo some positive or negative change when placed in a state of interaction with the disease-associated factors.

In the present specification, "level" of follicular T cells refers to the degree of the function of the T cells, and "amount" of follicular T cells refers to the degree of the physical amount of the T cells.

The term "viral antigen" as used herein refers to a part or all of a virus to be an object to be immunoreacted, and refers to a substance that causes an immune response when administered to a host. Peptides or whole (lysate) etc. can be used. Alternatively, virus-like particles (VLPs) may be formed and utilized. Examples of the virus antigen include, but are not limited to, antigens of HIV, hepatitis b virus, hepatitis c virus, coronavirus, norovirus, rotavirus, rabies virus, west nile virus, papilloma virus, zika virus, rubella virus, cytomegalovirus, influenza virus, and avian influenza virus.

In the present specification "bacterial antigen" means: some or all of the bacteria to be immunoreactive are substances which cause an immune response when administered to a host. Peptides or whole (lysate) etc. can be used. Examples of bacterial antigens include, but are not limited to, those derived from Bacillus anthracis (bacillus anthracis), bordetella pertussis (bordetella pertussis), borrelia burgdorferi (borrelia burgdorferi), brucella abortus (Brucella abortus), brucella canis (Brucella canis), brucella melitensis (Brucella caprae), brucella suis (Brucella suis), burkholderia mallei (burkholderia melitensis), burkholderia pseudomallei (burkholderia melitensis), campylobacter jejuni (campylobacter jejuni), chlamydia pneumoniae (chlamydia pneumoniae), chlamydia trachomatis (chlamydia trachomatis), chlamydophila psittaci (chlamydia psittaci), clostridium botulinum (clostridium botulinum), clostridium difficile (clostridium difficile), clostridium perfringens (clostridium perfringens), clostridium tetani (clostridium tetani), corynebacterium diptheriae (diphtheria), enterococcus faecalis (enterococcus), enterococcus faecium (enterococcus faecium), escherichia coli (Escherichia coli), enterotoxigenic Escherichia coli (Escherichia coli), enteropathogenic Escherichia coli (Escherichia coli), and Escherichia coli (Escherichia coli) and Escherichia coli (h157). H7 Antigens of Francisella tularensis (Francisella tularensis), haemophilus influenza (haemophilus influenzae), helicobacter pylori (helicobacter pylori), legionella pneumophila (Legionella pneumophila), leptospira interrogans (Leptospira interrogans), listeria monocytogenes (Listeria), mycobacterium leprae (leptospirillum), mycobacterium tuberculosis (Mycobacterium tuberculosis), mycoplasma pneumoniae (Mycoplasma pneumoniae), neisseria gonorrhoeae (Leptococcus), neisseria meningitides (Neisseria meningitides), pseudomonas aeruginosa (Pseudomonas aeruginosa), rickettsia rickettsia (Rickettsia), salmonella typhi (typhoid), salmonella typhimurium (Salmonella typhi), shigella Shigella sonnei), staphylococcus aureus (Staphylococcus aureus), staphylococcus epidermidis (Staphylococcus epidermidis), staphylococcus saprophyticus (Staphylococcus saprophyticus), streptococcus agalactiae (Streptococcus agalactis), streptococcus pneumoniae (Streptococcus pneumoniae), streptococcus pyogenes (Streptococcus pyogenes), treponema pallidum (Mycobacterium sypii), vibrio chlerae (Vibrio cholerae).

In the present specification "fungal antigen" means: part or all of the fungus to be immunoreactive is a substance which causes an immune response when administered to a host. Peptides or whole (lysate) etc. can be used. Examples of fungal antigens include, but are not limited to, antigens derived from Aspergillus clavatus (aspergillus clavatus), aspergillus flavus (aspergillus flavus), aspergillus fumigatus (aspergillus fumigatus), aspergillus nidulans (aspergillus nidulans), aspergillus niger (aspergillus niger), aspergillus terreus (aspergillus terreus), blastomyces dermatitidis (blastodermatitidis), candida albicans (Candida albicans), candida dubliniensis (Candida duvetica), candida glabra (Candida glabra), candida parapsilosis (Candida parapsilosis), candida rugosa (Candida rugosa), candida tropicalis (Candida tropicalis), cryptococcus albidus (cryptococcus gracilis), cryptococcus gattii (cryptococcus garvieae), cryptococcus laurentii (cryptococcus laurentii), cryptococcus neoformans (cryptococcus neoformans), histoplasma capsulatum (histoplasma capsulatum), microsporum canis (microsporomyces canis), pneumocystis carinii (pneumosporidium kaki), pneumocystis jirovecii (pneumosporidium cruzi), sporothrix schenckii (Candida crura), stachbotrys chartarum (staphylococcus, tina barbie, tinctoria (Tinea), tinctoria (trichia pastoris), and trichia pastoris (tie-back) and (tie-up).

In this specification "immune abnormality" means: any disease, disorder or condition that is generated or suspected to be generated due, at least in part, to an abnormality of the immune system. Refers to a state in which immunity is abnormal for some reason, and becomes susceptible to infection or allergic reaction. Examples of immune abnormalities include, but are not limited to, allergies, autoimmune diseases, and the like. In the case of self-derived antigens, autoimmune diseases are usually called, and exogenous antigens are called allergy. When the immune response is strong, the antigen from the self can be in an autoimmune disease state, and the non-self antigen can be in an allergic state; on the other hand, when the immune response is weak, the cancer state may be caused by self-antigens, and the infection may be caused by non-self-antigens.

The term "infection" as used herein may be any type of infection including viral infection (including any viral form such as single-or double-stranded DNA virus and RNA virus), bacterial infection, protozoal infection, and mycoplasma infection, and may be, for example: tuberculosis, coronavirus, malaria, yellow fever virus, smallpox virus, vaccinia, measles/rubella, polio, MUMPS (MUMPS)/MUMPS, rotavirus infection, varicella, yellow fever, ebola, west nile fever, haemophilus influenzae type B (Hib) infection, pneumococcal infection, pertussis, japanese encephalitis, neisseria meningitidis infection, salmonella infection, pathogenic escherichia coli, toxoplasma, zika virus, herpes virus type 1, EBV/epstein-barr virus (herpes virus type 4), CMV/cytomegalovirus (herpes virus type 5), influenza, MARS, rabies, diphtheria, and the like.

In the present specification, the term "cancer" is used in the same sense as commonly used in the art, and means: the atypical is strong, and it is more rapid than normal cell proliferation, and it can destructively infiltrate surrounding tissues or cause the existence of malignant tumor or similar malignant tumor. In the present invention, cancers include, but are not limited to, solid cancers and hematopoietic tumors, and "neoplasms" and "tumors" are used interchangeably herein. Cancers, neoplasms, tumors include not only solid neoplasms, but also neoplasms of the hematopoietic system. Examples of neoplasms include, but are not limited to: melanoma, non-small cell lung, hepatoma, retinoblastoma, astrocytoma, glioblastoma, gum, tongue, leukemia, neuroblastoma, head, neck, chest, pancreas, prostate, kidney, bone, testis, ovary, mesothelioma, sarcoma, cervix, gastrointestinal tract, lymphoma, brain, colon, bladder, myeloma, or other malignant or benign neoplasm. More specifically, the neoplasm may also be a hematopoietic neoplasm selected from the group consisting of acute myelogenous leukemia, acute lymphoblastic leukemia, myelodysplastic syndrome, chronic myelomonocytic leukemia, juvenile myelomonocytic leukemia, multiple myeloma, and chronic lymphocytic leukemia.

In the present specification, "autoimmune disease" means: autoantibodies (e.g., anti-Sm antibodies, anti-dsDNA antibodies, rheumatism factors) to self-components (e.g., immunoglobulins, etc.), autoimmune-related diseases are detected. Examples of autoimmune diseases include, but are not limited to: organ-specific autoimmune diseases (e.g., chronic thyroiditis, primary mucosal edema, thyrotoxicosis, pernicious anemia, goodpasture's syndrome (Goodpasture syndrome), acute progressive glomerulonephritis, myasthenia gravis, pemphigus vulgaris, bullous pemphigoid, insulin resistant diabetes mellitus, juvenile diabetes, addison's disease, atrophic gastritis, male infertility, early-onset menopause, lens-derived uveitis, sympathositism, multiple sclerosis, ulcerative colitis, primary biliary cirrhosis, chronic active hepatitis, autoimmune hemolytic anemia, narcolepsy, idiopathic thrombocytopenic purpura, and sjogren's syndrome, etc.), and organ-non-specific autoimmune diseases (e.g., rheumatoid arthritis, systemic Lupus Erythematosus (SLE), discoid lupus erythematosus, polymyositis, scleroderma, mixed connective tissue diseases, etc.). Rheumatoid arthritis, systemic lupus erythematosus, discoid lupus erythematosus, polymyositis, scleroderma and mixed connective tissue diseases have been known as collagenous diseases. That is, collagenous diseases are included in systemic autoimmune diseases.

In this specification "allergy" means: an excessive immune response to a specific non-self antigen is a disease that immunoreacts with an "allergen". "allergen" means: examples of the antigen capable of reacting with an antibody of a subject suffering from an allergic disease include, but are not limited to: allergens from tree pollen (acacia, alder, white wax (Fraxinus lanuginosa f. Velnna), beech, white birch, maple, huperzia serrata, red cedar, aspen, cypress, elm, yellow fir, rubber tree, eucalyptus, hackberry, hickory, yew, acer, mesquite, paper mulberry, oak, olive, hickory, pepper, pine, water tree, russian olive, phoenix tree, ailanthus altissima, black walnut, black willow, etc.), allergens from grass pollen (cotton, bermuda grass, kentucky, blue grass, sparrow, corn, cow tail grass, cogongrass, wild oat, duck grass, small furfuryl, ryegrass, rice, yellow crotalaria, timoth grass, amaranth, quinoa, siberian, etc.), and methods of preparing a plant pollen from plant pollen in particular, the present invention relates to a method for producing a plant, which comprises the steps of, in particular, solidago, belvedere, white chenopodium, calendula, nettle, barnyard grass, plantain, ragweed, perennial ragweed, wind-weed, artemisia rupestris, cytisine, sorrel, etc.), an insect-derived allergen (silkworm, mite, bee, wasp, ant, cockroach, etc.), a fungus-derived allergen (alternaria, aspergillus, botulinum, candida, cephalosporium, curvularia, epicoccum, epidermidis, fusarium, vermicularia, cladosporium, penicillium, phoma, pullulans (Pullularia pullulans), rhizopus, etc.), an animal hair-derived allergen (dog, cat, bird, etc.), a dust-derived allergen protein (House, etc.), a food-derived allergen (OVA, etc.), and the like. Representative diseases of "allergy" include atopic dermatitis, allergic rhinitis (pollinosis, etc.), allergic conjunctivitis, allergic gastroenteritis, bronchial asthma, pediatric asthma, food allergy, drug allergy, urticaria, etc.

In the present specification, "inflammatory disease" means: diseases or conditions characterized by abnormal inflammation (e.g., elevated levels of inflammation compared to a control in a healthy person or the like who is not afflicted with the disease). As non-limiting examples of inflammatory diseases, there may be mentioned: specific reactions, asthma, auto-inflammatory diseases, allergies, childhood allergic asthma, inflammatory bowel disease, celiac disease, crohn's disease, colitis, ulcerative colitis, collagenous colitis, lymphocytic colitis, diverticulitis, irritable bowel syndrome, short bowel syndrome, blind tab syndrome, chronic persistent diarrhea, infant refractory diarrhea, traveler's diarrhea, immunoproliferative small intestine disease, chronic prostatitis, post-enteritis syndrome, tropical stomatitis diarrhea, hueplerian disease, wolman's disease, arthritis, rheumatoid arthritis, behcet's disease, uveitis, pyoderma gangrenosum, erythema nodosum, traumatic brain injury psoriatic arthritis, juvenile idiopathic arthritis, multiple sclerosis, systemic Lupus Erythematosus (SLE), myasthenia gravis, juvenile onset diabetes, type 1 diabetes, gillin-barre syndrome, hashimoto's brain disease, hashimoto's thyroiditis, ankylosing spondylitis, psoriasis, sjogren's syndrome, vasculitis, glomerulonephritis, autoimmune thyroiditis, bullous pemphigoid, sarcoidosis, ichthyosis, graves ' eye disease, addison's disease, vitiligo, acne vulgaris, pelvic inflammatory disease, reperfusion injury, sarcoidosis, graft rejection, interstitial cystitis, atherosclerosis and atopic dermatitis.

HLA limitations should also be considered in the applicability of the techniques of the present invention. When cytotoxic (killer) T cells recognize a disease factor and are excluded by killing action, complexes of peptides from disease factor antigens recognized by the killer T cells with HLA class I molecules can be determined and such information can be used. Among the specific disease factors, there is a common HLA type possessed by a common antigen, and this method can be used to determine the antigenic peptides used in a specific population of the dendritic cell preparation of the present invention. When transgenic mice of this specific HLA class are immunized with these synthetic peptides, peptides that induce an immune response in killer T cells can be identified and utilized. Peptides Derived from antigens of disease factors binding to specific molecules of HLA having the highest frequency of occurrence in Japanese population can be determined by the same method as Tgm of HLA antigen, and the method can be used for determining antigenic peptides used in the specific population of the dendritic cell preparation of the present invention (the method can be referred to below: chen, Y.—Z., liu, G., senju, S..Wang, Q..Irie, A..Haruta, M.. … Nishimura, Y..2010). Identification of SARS-COV Spike Protein-modified and HLA-A2-Restricted Human CTL Epitopes by Using a New Muramyl Dipeptide-modified adhesive. International Journal of Immunopathology and Pharmacology,165-177.Https:// doi. Org/10.1177/039463201002300115). By using this information, it is possible to identify a viral antigen that binds to a specific HLA type (HLA type specific to a substance other than a specific substance, which is specific to a substance specific to a specific substance) recognized by killer T cells contributing to the elimination of disease factors, and if the dendritic cell vaccine of the present invention produced by using a differentiation induction method from human ES cells expressing these HLA is used, killer T cells can be activated antigen-specifically, and it is expected to prevent severe diseases caused by cytokine storm and the like. Representative antigenic peptides for use in the present invention may be HLA-restricted or HLA-non-restricted. If the peptide is limited, it can be handled by a single peptide or the like, and if the peptide is not limited, it is preferable to use a plurality of peptides overlapping each other or to electroporate a viral nucleic acid (electro corporation).

In addition, there are two types of T cells, cd8+ T cells and cd4+ T cells, when broadly classified. The antigens recognized by these two T cells are essentially different. Cd8+ T cells recognize peptides, typically consisting of 9 amino acids, that bind to HLA (MHC), a class 1 species. On the other hand, cd4+ T cells recognize peptides that bind to MHC called class II. The length of a peptide binding to HLA (MHC) class II is considered to be, for example, 15 to 24 (but not limited to) amino acids. HLA (MHC) class 1 molecules are expressed in all somatic cells and presented by binding to protein fragments produced by the own cells. HLA (MHC) class II molecules are expressed in antigen presenting cells such as macrophages and dendritic cells and in limited cells such as B cells. In general, unlike somatic cells, antigen-presenting cells acquire proteins from the outside, are decomposed, and then bind to HLA (MHC) class 1 and class II molecules for presentation. Thus, cd8+ T cells primarily recognize endogenous HLA (MHC) class 1 restricted antigens on somatic cells, and cd4+ T cells recognize exogenous HLA (MHC) class II restricted antigens on antigen presenting cells. In humans, MHC class 1 is HLA a, B, C, and MHC class II is HLA DR, DQ, DP.

The dosage form of the "drug (composition)" in the present specification is not particularly limited, and may be any of solid, semisolid, or liquid preparations, and may be selected depending on the purpose of use thereof, and the like. In the case of a cell preparation, the cell preparation is usually provided as a liquid preparation, but may be provided after freezing or freeze-drying. Examples of the dosage form of the pharmaceutical composition include those described in the seventeenth revised japanese pharmacopoeia general rule or the equivalent of each country.

In this specification "vaccine" means: a factor that comprises an antigen or a cell and is capable of producing an antibody when administered to an organism, a factor that enhances the ability of the cell to immunity, or a substance that is capable of producing the factor. The antigen refers to a selected substance, and refers to a composition for inducing an immune response against the substance in a vertebrate such as a human.

The terms "agent," "agent" or "factor" (all equivalent to those in english) are used interchangeably in a broad sense, and may be any substance or other element (e.g., light, radiant energy, heat, electricity, etc.) as long as the purpose of the figure is achieved. Examples of such substances include, but are not limited to: proteins, polypeptides, oligopeptides, peptides, polynucleotides, oligonucleotides, nucleic acids (e.g., including cDNA, DNA such as genomic DNA, RNA such as mRNA), polysaccharides, oligosaccharides, lipids, organic low molecules (e.g., hormones, ligands, information transfer substances, organic low molecules, molecules synthesized by combinatorial chemistry, low molecules useful as drugs (e.g., low molecular ligands, etc.), and the like.

In this specification "treatment" means: cure or ameliorate diseases or symptoms, or inhibit symptoms. In this state, the prevention of deterioration of such a disease or disorder is preferable, the maintenance of the present state is preferable, the alleviation is more preferable, and the resolution is further preferable, and the symptom-improving effect or the preventive effect of 1 or more symptoms associated with the disease or the accompanying disease of the patient can be exhibited. The diagnosis in advance and the appropriate treatment are called "concomitant treatment" or "customized treatment", and the diagnostic agent used for this purpose is sometimes called "concomitant diagnostic agent".

In this specification, "prevention" means: preventing before the disease or symptom appears.

In this specification, "treatment" means: any treatment of a disease or condition, including both therapeutic and prophylactic.

In this specification "diagnosis" means: various parameters related to a disease, disorder, status (e.g., cancer, viral infection, etc.) and the like of a subject are determined, and the present state or future of such disease, disorder, status is determined. By using the method, device and system of the present invention, the condition in the body can be checked, and using such information, various parameters such as the disease, disorder, and condition of the subject, and the prescription and method for treatment or prevention to be administered can be selected. In the present specification, the term "diagnosis" in a narrow sense means diagnosis of the present situation, and includes "early diagnosis", "predictive diagnosis", "pre-diagnosis", and the like in a broad sense. In principle, the diagnostic method of the present invention can be industrially useful because it can use a substance coming out of the body and can be carried out by a medical practitioner such as a doctor. In the present specification, "predictive diagnosis, advanced diagnosis or diagnosis" may be referred to as "support" in particular, for the sake of clarity, without being carried out by a medical practitioner such as a doctor.

In the present specification, "subject (person)" means: the object to be diagnosed, detected, treated, or the like of the present invention (for example, a living organism such as a human or cells, blood, serum, or the like extracted from the living organism).

In the present specification, "sample" means: any substance obtained from a subject or the like includes, for example, serum. Those skilled in the art can appropriately select a preferable sample according to the description of the present invention.

In this specification "kit" means: units of parts (e.g., examination drugs, diagnostic drugs, therapeutic drugs, antibodies, logos, instructions, etc.) to be provided are usually provided in more than 2 parts. Such a kit form is preferred when it is intended to provide, preferably, such a composition which is mixed prior to use and then used, in order to ensure stability and the like, which should not be mixed. Such a kit is preferably provided with instructions or instructions for the part provided (e.g. how to use the examination, diagnostic, therapeutic, or where the treatment should be). When the kit in the present specification is used as a kit (reagent kit), the kit generally contains instructions or the like describing methods of using a test agent, a diagnostic agent, a therapeutic agent, an antibody or the like.

In the present specification, "instruction" means: materials describing instructions for using the methods of the present invention are presented to a physician or other user. The instructions describe the detection method of the present invention, the method of using the diagnostic agent, or words and phrases indicating the content of administration. Further, the instruction may be written with words or phrases indicating the administration site such as oral administration, esophageal administration (for example, by injection, etc.). In this specification, the regulatory authorities of the country in which the present invention is implemented (for example, the ministry of labor for thick students in japan, the Food and Drug Administration (FDA) in the united states, etc.) are made in a format prescribed by, and are clearly documented as being approved by, their regulatory authorities. The directions are so-called page inserts (page inserts) and are usually provided in a paper medium, but are not limited thereto, and may be provided in the form of an electronic medium (e.g., a homepage or an email provided via the internet) or the like.

In this specification "functional equivalent" means: for the object entity, the target functions are the same but the structure is different. Thus, it should be understood that: a "disease-related factor" or a functional equivalent of a portion thereof, although not a disease-related factor or a portion thereof itself, variants or alterations (e.g., nucleic acid analogs, amino acid sequence alterations, etc.) of a disease-related factor or a portion thereof may become substances having biological effects possessed by a disease-related factor or a portion thereof, and at the time of action, may become variants or alterations of a disease-related factor or a portion thereof itself or a portion thereof (e.g., nucleic acids encoding variants or alterations of a disease-related factor or a portion thereof itself or a portion thereof, and include vectors, viruses, liposomes, cells, etc.) comprising nucleic acids. In the present invention, a functional equivalent of a disease-related factor or a part thereof is understood to be the same as the disease-related factor or a part thereof, even if not specifically described. Functional equivalents may be found by database retrieval. In this specification, "retrieving" means: other nucleobase sequences having particular functionalities and/or properties are found by electronic or biological or other methods using certain nucleobase sequences. The electronic search may be, but is not limited to: BLAST (Altschul et al, J.Mol.biol.215:403-410 (1990)), FASTA (Pearson & Lipman, proc.Natl.Acad.Sci., USA 85:2444-2448 (1988)), smith and Waterman (Smith and Waterman, J.Mol.biol.147:195-197 (1981)), and Needleman and Wunsch (Needleman and Wunsch, J.Mol.biol.48:443-453 (1970)), and the like. Biological searches may be exemplified by, but are not limited to: stringent hybridization (stringent hybridization), microarray (micro array) in which genomic DNA is attached to a nylon membrane or the like, microarray (micro array assay) in which genomic DNA is attached to a glass plate, PCR, in situ hybridization, or the like. In the present specification, the genes used in the present invention also include the corresponding genes identified by the above-mentioned electronic search and biological search.

In the present specification, "protein", "polypeptide", "oligopeptide" and "peptide" are used in the same sense in the present specification, and refer to a polymer of amino acids of any length. The polymer may be linear or branched, or cyclic. The amino acid may be a natural amino acid, an unnatural amino acid, or an altered amino acid. The term may also include substances that assemble on a complex of multiple polypeptide chains. The term also includes naturally or artificially altered amino acid polymers. Such alterations include, for example, thioether bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or alteration (e.g., conjugation to a labeling element). Also included in this definition are, for example, polypeptides comprising analogs of 1 or more than 2 amino acids (e.g., comprising unnatural amino acids, etc.), peptide-like compounds (e.g., peptides), and other variations known in the art. In the present specification, the term "amino acid" refers to a generic term for organic compounds having an amino group and a carboxyl group. When an antibody of an embodiment of the present invention comprises a "specific amino acid sequence," any one of the amino acids in the amino acid sequence may be chemically modified. In addition, any one of the amino acids in the amino acid sequence may also form a salt or solvate. In addition, any one of the amino acids in the amino acid sequence may be L-form or D-form. In these cases, it can be said that the protein according to the embodiment of the present invention contains the above-described "specific amino acid sequence". As chemical modifications to be applied to amino acids contained in proteins in vivo, for example, N-terminal modifications (for example, acylation, myristoylation, etc.), C-terminal modifications (for example, amidation, glycosyl phosphatidylinositol addition, etc.), side chain modifications (for example, phosphorylation, sugar chain addition, etc.), and the like are known. Amino acids may be naturally or non-naturally occurring as long as the objects of the present invention are met.

In the present specification, "polynucleotide", "oligonucleotide" and "nucleic acid" are used in the same sense in the present specification, and refer to a polymer of nucleotides of arbitrary length. The term also includes "oligonucleotide derivatives" or "polynucleotide derivatives". "oligonucleotide derivative" or "polynucleotide derivative" refers to: including derivatives of nucleotides, or linkages between nucleotides, are used interchangeably with generally different oligonucleotides or polynucleotides. Examples of such an oligonucleotide include a 2' -O-methyl-ribonucleotide, an oligonucleotide derivative in which a phosphodiester bond in the oligonucleotide is converted to a phosphorothioate bond, an oligonucleotide derivative in which a phosphodiester bond in the oligonucleotide is converted to an N3' -P5' -phosphoramide bond, an oligonucleotide derivative in which ribose and a phosphodiester bond in the oligonucleotide are converted to peptide nucleic acid bonds, an oligonucleotide derivative in which uracil in the oligonucleotide is substituted with C-5-propargyl uracil, an oligonucleotide derivative in which uracil in the oligonucleotide is substituted with C-5-thiazolyl uracil, an oligonucleotide derivative in which cytosine in the oligonucleotide is substituted with C-5-propargyl cytosine, an oligonucleotide derivative in which cytosine in the oligonucleotide is substituted with phenoxazine (phenoxazine-modified cytosine), an oligonucleotide derivative in which ribose in DMA is substituted with 2' -O-propyl, and an oligonucleotide derivative in which ribose in the oligonucleotide is substituted with 2' -methoxyethoxy. Unless otherwise indicated, a particular nucleic acid sequence, as well as the indicated sequence, comprises conservatively modified alterations (e.g., degenerate codon substitutions) and complementary sequences thereof. Specifically, degenerate codon substitutions can be achieved by making a sequence in which the 3 rd position of 1 or more selected (or all) codons is substituted with mixed base and/or deoxyinosine residues (Batzer et al, nucleic Acid Res.19:5081 (1991); ohtsuka et al, J.biol.chem.260:2605-2608 (1985); rossolini et al, mol.cell.probes 8:91-98 (1994)). "nucleic acid" in this specification is also used interchangeably with gene, cDNA, mRNA, oligonucleotide, and polynucleotide. "nucleotides" in this specification may be natural or unnatural.

In the present specification, "gene" means: the factors that determine the genetic trait, "gene" sometimes refers to "polynucleotide", "oligonucleotide" and "nucleic acid".

"identity" of genes in this specification means: the degree of identity of 2 or more gene sequences with respect to each other, generally has "identity" means: the degree of identity or similarity is high. Thus, the higher the identity of a given 2 genes, the higher the identity or similarity of their sequences. Whether 2 genes have identity can be checked by direct comparison of the sequences or, in the case of nucleic acids, by hybridization under stringent conditions. When 2 gene sequences are directly compared, typically at least 50% identical, preferably at least 70% identical, more preferably at least 80%, 90%, 95%, 96%, 97%, 98% or 99% identical, in the DNA sequence between the gene sequences. Accordingly, the term "identical body" or "identical gene product" in the present specification means another protein of another species that exhibits the same biological function as the protein constituent element of the complex described further in the present specification, and preferably means a mammalian protein. Such isoforms are sometimes also referred to as "orthologous gene products". It is understood that such homologues, identical gene products, orthologous gene products, etc. may also be used as long as they meet the objectives of the present invention.

In the present specification, amino acids may be represented by commonly known 3-letter symbols or single-letter symbols recommended according to IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides can likewise be represented by commonly recognized single-letter symbols. In the present specification, the similarity, identity and identity between amino acid sequences and base sequences can be calculated by using BLAST, a tool for sequence analysis, and default parameters. The identity can be retrieved, for example, using NCBI's BLAST 2.2.28 (which may be 2013.4.2 published or updated). The value of identity in the present specification generally refers to the value corrected by default using BLAST described above. However, when a higher value is obtained by changing the parameter, the maximum value is taken as the value of the identity. When evaluating identity in a plurality of fields, the maximum value thereof is taken as the value of identity. Similarity is a numerical value calculated for similar amino acids in addition to identity.

In one embodiment of the present invention, "plurality" means: for example, 8, 7, 6, 5, 4, 3, or 2 may be used, or any one of these values may be used. Polypeptides having deletions, additions, insertions, or substitutions of 1 or more amino acid residues with other amino Acids are known to maintain their biological activity (Mark et al, proc Natl Acad Sci USA.1984Sep;81 (18): 5662-5666, zoller et al, nucleic Acids Res.1982Oct 25;10 (20): 6487-6500, wang et al, science.1984Jun 29;224 (4656): 1431-1433.). By measuring the activity by a suitable method such as deletion, it is possible to determine whether it is a functional equivalent.

In one embodiment of the present invention, "90% or more" means: for example, 90, 95, 96, 97, 98, 99, or 100% or more may be used, or any 2 values may be used. The above "identity" is the ratio of the number of identical amino acids in 2 or more amino acid sequences that can be calculated according to methods well known in the art. Before calculating the determined ratio, the amino acid sequences of the compared sets of amino acid sequences are aligned, and if it is desired to maximize the ratio of identical amino acids, gaps are introduced in a portion of the amino acid sequences. Methods for alignment, methods for calculation of scale, methods for comparison, and these related computer programs are well known in the art (e.g., BLAST, GENETYX, etc.). "identity" in the present specification can be expressed by a value measured by BLAST of NCBI unless otherwise specified. Blastp can be used by default by BLAST algorithm when comparing amino acid sequences. The measurement results were quantified as posives or ids.

The substance of the present invention may be a purified substance, and "purified" substance or biological factor (e.g., nucleic acid or protein, etc.) in the present specification means: a substance from which at least a portion of the substance naturally associated with the substance or biological factor has been removed. Thus, in general, the purity of a biological factor of the purified biological factors is higher (i.e., concentrated) than the state in which the biological factor is normally present. The term "purified" as used in the present specification means: the same type of biological factor is present, preferably at least 75 wt%, more preferably at least 85 wt%, still more preferably at least 95 wt%, most preferably at least 98 wt%. The substances or biological factors used in the present invention are preferably "purified" substances. As used herein, "isolated" material or biological factor (e.g., nucleic acid or protein, etc.) refers to: a substance from which a substance naturally accompanying the substance or biological factor has been substantially removed. The term "isolated" as used in this specification varies depending on its purpose and is therefore not necessarily expressed in terms of purity, but means that there is preferably at least 75% by weight, more preferably at least 85% by weight, still more preferably at least 95% by weight, most preferably at least 98% by weight of the same type of biological factor, if necessary. The material used in the present invention is preferably a "isolated" material or biological factor.

In this specification "corresponding" amino acid or nucleic acid or moiety means: a polypeptide molecule or polynucleotide molecule (e.g., a polynucleotide encoding a spike protein) has the same action as or is predicted to have the same action as a predetermined amino acid or nucleotide or part of a polypeptide or polynucleotide to be used as a reference for comparison, and particularly an enzyme molecule refers to an amino acid that is present at the same position in the active site and contributes to the same activity of a catalyst, and a complex molecule refers to a corresponding part (e.g., heparin sulfate). For example, if an antisense (antisense) molecule is used, the antisense molecule may be the same part of an orthologous gene corresponding to a specific part of the antisense molecule. The corresponding amino acid may be, for example, a specific amino acid subjected to cysteinylation, glutathionylation, S-S bond formation, oxidation (for example, oxidation of methionine side chain), formylation, acetylation, phosphorylation, sugar chain addition, myristoylation, or the like. Alternatively, the corresponding amino acid may be an amino acid responsible for dimerization. Such "corresponding" amino acids or nucleic acids may be over a range of regions or areas (domains). Therefore, in this case, it is referred to as a "corresponding" region or zone in this specification. Such corresponding regions or zones are useful in designing the composite molecules in this specification.

In this specification, "corresponding" gene (e.g., polynucleotide sequence or molecule) refers to: in a species, a gene (e.g., a polynucleotide sequence or a molecule) that has the same effect as a predetermined gene in the species to be the reference for comparison, or that is predicted to have the same effect, when a plurality of factors having such an effect exist, refers to a gene having the same origin as the chemical process. Thus, a gene corresponding to a gene may be ortholog (ortholog) of the gene. Thus, the specific genes of humans can be found in other animals (in particular, mammals), respectively. Such corresponding genes can be determined using techniques well known in the art. Thus, for example, a gene (e.g., S protein) corresponding to a gene corresponding to an animal (e.g., mouse) can be found by searching a database containing the sequences of the animal using the sequence of the gene corresponding to the gene as a query sequence.

In this specification "fragment" means: for full-length polypeptides or polynucleotides (length n), polypeptides or polynucleotides having a sequence length ranging from 1 to n-1 are provided. The length of the fragment may be appropriately changed depending on the purpose, for example, the lower limit of the length, and in the case of a polypeptide, amino acids of 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50 and more are exemplified, and the length (for example, 11 and the like) represented by an integer not specifically exemplified herein may be suitable as the lower limit. In the case of polynucleotides, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100 or more nucleotides are exemplified. Here, a length (for example, 11 or the like) represented by an integer not specifically recited may be suitable as the lower limit. In this specification, such fragments, for example when a full length substance functions as a marker or target molecule, are to be understood as also falling within the scope of the invention as long as the fragment itself also functions as a marker or target molecule.

According to the present invention, the term "activity" is used in the present specification to mean: the function of the molecule in its broadest sense can be referred to in the evaluation of functional equivalents. The activity is not particularly limited, but generally includes a biological function, a biochemical function, a physical function, or a chemical function of a molecule. Activity includes, for example, enzymatic activity, the ability to interact with other molecules, and the ability to activate, promote, stabilize, hinder, inhibit or destabilize the function of other molecules, stability, the ability to exist locally at a specific intracellular location. Where applicable, this term also relates to the function of the protein complex in its broadest sense.

In the present specification, "biological function" means: when referring to a gene or its related nucleic acid molecule or polypeptide, the gene, nucleic acid molecule or polypeptide may have a specific function in an organism. Examples thereof include, but are not limited to: specific antibody production, enzyme activity, resistance, and the like. In the present specification, a biological function can be exerted by "biological activity". In the present specification, "biological activity" means: the activity that a factor (e.g., polynucleotide, protein, etc.) has in an organism includes activity that performs various functions (e.g., the ability to trigger follicular T cells), such as activity that also includes activation or inactivation of other molecules by interaction with a molecule. When 2 factors interact, the biological activity may be the bond between two molecules and the biological change it causes, and for example, when 1 molecule is precipitated using an antibody, other molecules are co-precipitated, and then 2 molecules are considered to bind to each other. Thus, observing such co-precipitation is one judgment method that can be enumerated. For example, where a factor is an enzyme, its biological activity includes its enzymatic activity. In other examples, where a factor is a ligand, this includes binding of the ligand to its corresponding receptor. Such biological activity can be determined by techniques well known in the art. Thus, "activity" refers to: presentation or shows a combination (either direct or indirect); various measurable indicators of the effect (i.e., having a measurable effect on the response to certain exposures or stimuli) of the response may be exemplified by the affinity of the compound to which the polypeptide or polynucleotide of the invention binds directly, or by the amount of protein upstream or downstream after various certain stimuli or events, or other similar functional scales.

In the present specification, "expression" of a gene, polypeptide, polynucleotide, etc. means: the gene and the like are transformed into other forms after being acted on a certain degree in the organism. Preferably, it is: genes, polynucleotides, and the like are transcribed or translated and converted into a polypeptide form, but are also expressed as mRNA after transcription. Thus, in this specification "expression product" means: including such polypeptides or proteins or mRNA. More preferably, the form of such polypeptide may be one after post-translational treatment (processing).

As functional equivalents of the present invention, use may be made of products in which 1 or more amino acids in the amino acid sequence are inserted, substituted or deleted, or added at one or both ends thereof. In the present specification, "1 or more amino acid insertions, substitutions or deletions in an amino acid sequence, or additions at one or both ends thereof" means: the modification may be performed by a known technique such as site-specific mutagenesis or by substitution of a plurality of amino acids to such an extent that natural mutation can be naturally produced. The modified amino acid sequence may be, for example, an insertion, substitution or deletion of 1 to 30, preferably 1 to 20, more preferably 1 to 9, still more preferably 1 to 5, particularly preferably 1 to 2 amino acids, or a product obtained by adding the amino acids to one or both of the termini. The amino acid sequence of the modified amino acid sequence is preferably an amino acid sequence having 1 or more (preferably 1 or more or 1, 2, 3 or 4) conservative substitutions in the amino acid sequence of the S protein or the like of the virus. Herein, "conservative substitution" refers to: to replace 1 or more amino acid residues with other chemically similar amino acid residues without substantially altering the function of the protein. For example, a case where a certain hydrophobic residue is substituted with another hydrophobic residue, a case where a certain polar residue is substituted with another polar residue having the same charge, and the like can be exemplified. Among functionally similar amino acids for each amino acid that can undergo such substitution, each amino acid is well known in the art. Specific examples of the nonpolar (hydrophobic) amino acid include alanine, valine, isoleucine, leucine, proline, tryptophan, phenylalanine, methionine and the like. Examples of polar (neutral) amino acids include glycine, serine, threonine, tyrosine, glutamine, asparagine, and cysteine. Examples of the positively charged (basic) amino acid include arginine, histidine, and lysine. Amino acids having negative charges (acidity) include aspartic acid, glutamic acid, and the like.

(preferred embodiment)

Preferred embodiments of the present invention will be described below. The following examples are provided for a better understanding of the present invention, and the scope of the present invention should not be limited to the following. Accordingly, it will be apparent to those skilled in the art from this disclosure that appropriate modifications may be made within the scope of the invention. In addition, the following embodiments of the present invention may be used alone or in combination.

The present invention relates to a method for evaluating a disease in a subject, and a composition, a system, a program for storing the same, and any other medical technique related thereto, which comprises a step of measuring the level or amount of follicular T cells reactive with a factor related to the disease in the subject.

In the present invention, the medical technique of the present invention further comprises a step of performing at least 1 selected from the group consisting of evaluation of the history of the disease, evaluation of the effectiveness of a prophylactic or vaccine for the disease, evaluation of the effectiveness of a therapeutic agent for the disease, evaluation of the ability to defend against the disease again, evaluation of the disease condition and evaluation of the risk of developing the disease, based on the measurement result of the level or amount of follicular T cells reactive to factors related to the disease.

In one embodiment, these evaluations include, but are not limited to: for example, evaluation of infection history, evaluation of vaccine effectiveness (whether Tfh induction is present or not), evaluation of reinfection defense ability, evaluation of effectiveness of a cancer immune drug (immune checkpoint or the like), evaluation of risk of autoimmune disease accompanied by autoantibodies (when not diseased), evaluation of disease condition, and the like.

In one aspect, the present invention provides a method of producing follicular T cells specific for a disease, the method comprising a step of determining a disease-related factor or a part thereof having the ability to elicit follicular T cells specific for a disease-related factor of the disease, a step of eliciting follicular T cells specific for the disease using the disease-related factor or part thereof, and a step of obtaining follicular T cells specific for the disease.

In one aspect, the invention provides a method of screening for follicular T cells reactive with a disease-related factor, the method comprising: a) providing a follicular T cell mass, B) bringing the follicular T cell mass into contact with a disease-related factor, C) measuring the reactivity of the follicular T cell with the disease-related factor or a part or functional equivalent thereof, D) selecting, from the follicular T cell mass, a follicular T cell exhibiting a reactivity of a predetermined value or more with respect to the disease-related factor.

In one embodiment, the step of a) providing the follicular T cell mass may be performed by blood collection or biopsy from a subject.

In one embodiment, the step of B) contacting the follicular T cell mass with a disease-related factor or a portion or functional equivalent thereof may be performed by: for example, a disease-related factor or a part or functional equivalent thereof is added to a culture solution in which a follicular T cell group is cultured, and thereafter, if necessary, culture, incubation, or standing is performed.

In one embodiment, the step of C) determining the reactivity of the follicular T cells to the disease-associated factor or a portion or functional equivalent thereof may be performed by ELISPOT, flow cytometry, re-stimulation of Peripheral Blood Mononuclear Cells (PBMCs), new generation sequencer/genetic testing (histology), immunostaining (pathology), in situ analysis.

In one embodiment, the prescribed value may be an average or median of the reactivity of follicular T cells in a non-diseased subject to a disease-related factor or a portion or functional equivalent thereof.

< diagnosis >

In one embodiment, the invention provides a method for the examination or diagnosis of a disease comprising the step of determining the level or amount of follicular T cells reactive with a disease related factor.

In another aspect, the invention provides a method for testing a subject for a disease, the method comprising the steps of determining the level or amount of follicular T cells reactive with a disease-related factor of the disease in the subject, and comparing the level or amount to a specified baseline. In some embodiments, the prescribed reference may be an average or median level or amount of follicular T cells in a healthy person. In other embodiments, the specified benchmark may be a reference value for the paper.

In another aspect, the present invention provides a method for evaluating the infection history of a disease, a method for evaluating the vaccine effectiveness of a disease, evaluating the reinfection defense capacity, a method for evaluating the effectiveness of a cancer immune drug (immune checkpoint, etc.) of a disease, or a method for evaluating the risk of autoimmune disease accompanied by autoantibodies (when not diseased), and evaluating the disease condition. These methods include the step of determining the level or amount of follicular T cells reactive with a disease-related factor.

In some embodiments, the above-described assays may also be performed by ELISPOT, flow cytometry, re-stimulation assays of Peripheral Blood Mononuclear Cells (PBMCs), new generation sequencer/genetic testing (panel analysis), immunostaining (pathological tissue inspection), in situ analysis.

In one embodiment, the determination of the level or amount of follicular T cells reactive with a disease-related factor may be performed, for example, as follows.

Peripheral blood from a subject may be contacted with a probe containing an epitope specific for the source of infection, and the amount of follicular T cells in the peripheral blood bound to the probe may be determined by ELISPOT or flow cytometry, as exemplified elsewhere in this specification.

ELISPOT used in the present invention may be used. In the ELISPOT assay used herein, plates coated with antibodies that detect IL-21 produced by follicular T cells are used. Peripheral Blood Mononuclear Cells (PBMCs) were added to the wells and stimulated with antigen from the infectious source for 36 hours. The secreted IL-21 binds to the capture antibody at the periphery of the producer cell. After removing cells by washing, a cytokine antibody for detection was added, and spots were detected. As a negative control, the assay was performed without stimulation with antigen from the infectious source.

Flow cytometry may be utilized in the present invention that uses antigens from an infectious source to stimulate Peripheral Blood Mononuclear Cells (PBMCs) for 36 hours. FCM analysis was performed for FSC SSC gag, PI negative gating, CD3 positive gag, double positive fractionation of CD4, CXCR5, and/or triple positive fractionation of HLA tetramers that further bind to antigen from the infectious source. Comparison was made with a sample as a negative control to which no stimulus of the antigen from the infectious source was applied.

In a specific embodiment, the disease can be examined or diagnosed by comparing the level or amount of follicular T cells measured as being reactive to a disease-related factor with a prescribed value (e.g., a negative control measurement or a healthy person measurement).

In another aspect, the invention provides a method for evaluating the infection history of a disease, the method comprising the step of determining the level or amount of follicular T cells reactive with a disease related factor. Although evaluation of infection history based on antibodies is often performed, specific immune cells are maintained longer than antibodies, and thus it is useful to determine specific immune cells of an infection source in terms of investigation of infection history.

In one embodiment of the evaluation of infection history of a disease, the measurement of the level or amount of follicular T cells reactive to a factor associated with the disease can be performed, for example, as follows.

In a specific embodiment, the infection history of the disease can be evaluated by comparing the level or amount of follicular T cells reactive to a disease-related factor with a predetermined value (e.g., a negative control measurement value or a healthy person measurement value).

In another aspect, the invention provides a method for evaluating vaccine effectiveness, evaluating reinfection defense capacity of a disease, the method comprising the step of measuring the level or quantity of follicular T cells reactive to a disease-related factor. Specific immune cells are maintained longer than antibodies, and the defenses of immune cells can be evaluated in terms of their ability to respond rapidly upon reinfection.

In one embodiment of determining the level or amount of follicular T cells reactive to a disease-related factor, the determination of the level or amount of follicular T cells reactive to a disease-related factor may be performed, for example, as follows.

In a specific embodiment, the vaccine effectiveness and reinfection protection against a disease can be evaluated by comparing the level or amount of follicular T cells measured as being reactive to a disease-related factor with a predetermined value (e.g., a negative control measurement or a healthy person measurement).

In another aspect, the invention provides a method for evaluating the effectiveness of a cancer immune drug (immune checkpoint, etc.) of a disease, the method comprising the step of determining the level or amount of follicular T cells reactive with a cancer-related factor.

In one embodiment of determining the level or amount of follicular T cells reactive to a cancer-related factor, the determination of the level or amount of follicular T cells reactive to a disease-related factor may be performed, for example, as follows.

In a specific embodiment, the effectiveness of a cancer immune drug (immune checkpoint, etc.) against a disease can be evaluated by comparing the level or amount of follicular T cells measured to be reactive to a disease-related factor with a predetermined value (e.g., a negative control measurement or a healthy person measurement).

In a specific aspect, the present invention provides a method for risk assessment (when not suffering from a disease) or disease assessment of autoimmune diseases accompanied by autoantibodies. Among the autoimmune diseases accompanied by autoantibodies, the presence or absence or increase or decrease of follicular T cells interacting with B cells producing autoantibodies among diseases considered to be pathogenic by autoantibodies (e.g., ANCA-related vasculitis) are considered to be involved in the onset and exacerbation/recurrence of diseases accompanied by maturation of B cells.

In one embodiment of determining the level or amount of follicular T cells reactive to an autoimmune disease-related factor, the determination of the level or amount of follicular T cells reactive to a disease-related factor can be performed, for example, as follows.

In a specific embodiment, the level or amount of the follicular T cells that are measured to be reactive to a disease-related factor is compared with a predetermined value (for example, a measurement value of a negative control or a measurement value of a healthy person), and thus the risk evaluation (when not suffering from a disease) of autoimmune diseases accompanied by autoantibodies and the disease condition can be evaluated.

In particular embodiments, diagnosis or examination may be performed by measuring the various amounts or levels described above. The diagnosis and examination can be applied to autoimmune diseases, other diseases (neurodegenerative diseases, etc.), and diseases in which the immune repertoire is changed, and specific examples are expected as described below.

1. Evaluation of infection history

2. Evaluation of vaccine effectiveness (whether Tfh Induction exists)

3. Evaluation of Reinfection defense ability

4. Evaluation of cancer immune drug (immune checkpoint etc.) effectiveness

5. Risk assessment (when not suffering from an autoimmune disease) and disease assessment associated with autoantibodies

In one embodiment, the present invention provides a technique for evaluating infection histories.

Although evaluation of infection history by antibodies is often performed, specific immune cells are maintained longer than antibodies, and thus it is useful to determine specific immune cells of an infection source in terms of investigation of infection history. The T cell assay using a probe containing an epitope specific to the infectious agent is useful as a method for easily detecting immune cells established and maintained after infection. The presence or absence of T cells that react with the probes can be readily detected by ELISPOT using Peripheral Blood Mononuclear Cells (PBMCs) or by a system using a flow cytometer. If a probe-reactive T cell is detected, it can be determined that there has been an infection in the past. In addition, if a larger number of probe-reactive T cells are detected than in healthy people, it can be determined that there has been an infection in the past.

Use for diagnosis of infection

Specific responses based on immune cells are known to be established for days to about 2 weeks after infection, and thereafter to be maintained for a long period of time. Therefore, if a selected probe is used, after infection, the infectious agent is removed from the body or suppressed to a state where it is undetectable, and the infection history can be determined from the immune state.

Specific order of evaluation of infection histories

The reactivity of follicular T cells is confirmed using an epitope associated with a specific infection and a test body in which the patient or restorer is considered to have established an acquired immunity to the specific infection, and epitopes inducing follicular T cells are determined. Here, in order to ensure that the sequence conservation of other infectious antigens or disease-associated antigens to an epitope is evaluated based on the response of immune cells specific to a particular infectious disease, it is desirable to actually evaluate cross-reactivity using a subject or epitope-reactive T cells, and select an epitope specific to a particular infectious disease of interest. This ensures that the epitope selected when there is only a history of infection for a specific infectious disease reacts, and can be used for evaluation of infection history.

In one embodiment, the invention provides an evaluation of vaccine effectiveness, an evaluation of reinfection protection capability.

Specific immune cells are maintained longer than antibodies, and the defenses of immune cells can be evaluated in terms of their ability to respond rapidly upon reinfection. In addition, since follicular T cells are important for induction and enhancement of humoral immunity (affinity maturation) among T cell fractionation (T cell fractionation), it can be determined that a subject having T cells that react with a probe containing an epitope of the infection source that induces follicular T cells has a stronger infection defense capability or a superior defense capability. Since the immune cell-based specific response only reacts to vaccine antigens, by observing the reactivity based on this epitope, the presence or absence of immunity and intensity against the disease of the vaccine subject after vaccination can be judged.

Specific order of evaluation of vaccine effectiveness and reinfection defense Capacity

Among T cell epitopes contained in the vaccine, the reactivity of follicular T cells is confirmed using a subject whose acquired immunity against the vaccine-subject disease is considered to be established in a patient or restorer of the vaccine-subject disease, and epitopes that induce follicular T cells are determined. Here, in order to ensure that the sequence conservation of other infectious antigens or disease-associated antigens to an epitope is evaluated based on the response of immune cells specific to a particular infectious disease, it is desirable to actually evaluate cross-reactivity using a subject or epitope-reactive T cells, and select an epitope specific to a particular infectious disease of interest. . This ensures that the vaccine will react with only the duration of infection for a particular infectious disease and the epitope selected in the vaccination, and be used for the evaluation of vaccine effectiveness. Since the immune cell-based specific response only reacts to vaccine antigens, by observing the reactivity based on this epitope, the presence or absence of immunity and intensity against the disease of the vaccine subject after vaccination can be judged. Thus, the vaccine effectiveness and the reinfection defense ability can be evaluated.

As described above, it is considered that: it is considered that it is effective to examine cells that are responsive to probes of PBMC as an immunoassay established after infection or after vaccine intake. The evaluation method may be performed by using ELISPOT or flow cytometry in the same manner as described above. These methods can be used to detect whether or not T cells that react with the epitope probe are actually follicular T cells by changing the cytokine to be detected or combining the surface antigen probes, and can design an experimental system based on the detection sensitivity or the judgment standard.

Follicular T cells are characterized by a surface antigen and released cytokines. Thus, in the case of ELISPOT, it is possible to confirm whether or not the reacted T cell is a follicular T cell by detecting a cytokine specific to the follicular T cell released from the cell, specifically IL-21 or the like, after the reaction with the epitope, and in the case of flow cytometry, it is possible to confirm whether or not the reacted T cell is a follicular T cell by capturing a cell surface antigen characteristic to the follicular T cell such as CXCR 5. Furthermore, by capturing cytokines or surface antigens unique to other T cells simultaneously, the dominance of follicular T cells in T cells induced by each epitope can be evaluated. Here, the released cytokine can be evaluated for the ability to induce T cells of the epitope-specific follicular type by comparing with the cytokine before the epitope stimulation or by comparing with a positive control/negative control prepared as a control in advance, and thus, based on the value, the immunity established after infection or after vaccination can be evaluated.

In other embodiments of the invention, the ability to defend against reinfection may be assessed.

The intensity of immunity against the target infectious disease maintained by immunological memory can be understood by detecting the vaccine antigen, or the response of infectious antigen specific follicular T cells by ELISPOT or flow cytometry as described above. There are cases where strict and extensive epidemiological studies are required, but it is possible to use the correlation between the level of response or the measured value of follicular T cells and infection defense or severe prophylaxis (the correlation between the number of people who evaluate the presence of infection and the level of response or the measured value of follicular T cells in a subject for a certain period of time) as an index of reinfection defense ability.

In one embodiment, the present invention provides an evaluation of the effectiveness of cancer immune drugs (immune checkpoints, etc.).

If a humoral immune response against a cancer antigen is elicited, a stronger antitumor property can be expected. Thus, more test subjects are present with follicular T cells that are induced to an anti-tumor humoral immune response, and the anti-tumor immunity is easily activated, and drugs such as immune checkpoint inhibitors that enhance the anti-tumor immunity of the test subjects are considered to be more effective.

Tumor-specific T cells of each patient are extremely small in peripheral blood and are difficult to detect in vitro (ex vivo), but the response to a tumor can be detected by a method of concentrating tumor-specific T cells by co-culture with a tumor of the patient or the like. Since it is expected that the response of cytotoxic (cd8+) T cells will be stronger, by removing cd8+ T cells from the periphery and co-culturing cd4+ T cells, antigen presenting cells (immortalized B cells, etc.) with a tumor of a patient or a tumor cell line derived from a patient, the response of tumor-specific follicular T cells can be easily observed, and the antitumor immunity of a subject can be evaluated based on the level of the response of follicular T cells or the measurement value. More preferably, follicular T cells present in a tumor local or tumor microenvironment can be evaluated by in vitro testing using a surgical or biopsy sample performed prior to administration of the cancer immune drug. Here, not only the amount of follicular T cells or the level of cytokines can be evaluated, but also the level of chemokines such as CXCL13, which are considered to induce follicular T cells in the future, and further the level of inhibitory cytokines such as IL-7, which are suggested to be correlated with a three-dimensional lymphoid structure with good correlation with prognosis, or IL-10, which are suggested to be correlated negatively, can be evaluated in combination, and thus, more accurate and effective evaluation can be performed.

Consider that: by evaluating the reactivity between T cells derived from a tumor biopsy sample or peripheral blood and an epitope probe, the activity of anti-tumor immunity of a subject can be evaluated, thereby contributing to the selection of a therapeutic method. Furthermore, in combination with an epitope probe and a flow cytometer, it is expected to examine whether or not antitumor immune activity is maintained by evaluating the activation or depletion degree of epitope-specific follicular T cells (tumor-specific T cells) at the time of administration. This is effective in determining whether or not an immune checkpoint inhibitor is effective in the future, such as whether or not the treatment should be discontinued and other treatment methods should be searched for.

Activation of each follicular T cell can be evaluated, for example, by expression of IL-21 or ICOS, and production performance of IL-21. Although the degree of depletion is generally assessed by PD-1, other depletion markers LAG3, TIM3, TIGIT, etc. may be used.

The method for evaluating the degree of depletion of T cells used in the present specification can be carried out according to any method known in the art. For example, the degree of depletion of T cells can be assessed by flow cytometry or the proportion of PD-1 positive cells (in fractionation of the cells of interest) in an equivalent assay. The degree of depletion may be 20% to 100%. Although not particularly limited, the degree of exhaustion can be measured according to the method described in "PD-1BlockadeTherapy Promotes Novel Infiltration of Tumor-Specific T-CellClonotypes" https:// papers. Ssrn. Com/sol3/papers, cfmabstract_id= 3796528).

In one embodiment, the present invention provides a risk assessment (when not suffering from an autoimmune disease) or a disease assessment of autoimmune diseases accompanied by autoantibodies.

There are many autoimmune diseases accompanied by autoantibodies, but among them, the presence or absence or increase or decrease of follicular T cells interacting with B cells producing autoantibodies in diseases considered to be pathogenic by autoantibodies (e.g., ANCA-related vasculitis) are considered to be associated with onset of maturation of B cells, exacerbation/recurrence of the disease. Thus, it is thought that by detecting follicular T cells presenting epitopes from disease-specific autoantigens, disease risk (pre-onset) or disease activity can be assessed.

Evaluation can be performed by detecting follicular T cells presenting epitopes derived from autoantibodies, which are the cause of autoimmune diseases. The assay method may consider a probe-reactive cytocheck of PBMCs sampled from a subject using ELISPOT or flow cytometry.

When an increase in follicular T cells is confirmed as compared with the negative control, it can be judged that the risk of disease (before onset) or the disease activity is high.

(method for producing antigen)

The polypeptide of the present invention can be produced by various methods such as chemical synthesis and methods for producing by microorganisms using genetic engineering.

Those skilled in the art will appreciate, in light of the present disclosure, that there are various methods of making antigens. In general, antigens may be made by either in vitro or in vivo. The antigen may be produced in vitro as a peptide or polypeptide, which is then produced as a personalized new vaccine or immunogenic composition and administered to a subject. As described in more detail in the present specification, the in vitro production can be performed by various methods known to those skilled in the art, such as expression of peptides/polypeptides derived from DNA or RNA molecules in peptide synthesis or any of various bacterial, eukaryotic, or viral recombinant expression systems, and purification of the continuously expressed peptides/polypeptides. Alternatively, the antigen may be produced in vivo by introducing an antigen into a subject as a molecule encoding the antigen (e.g., DNA, RNA, a viral expression system, etc.), and expressing the encoded antigen after the introduction. Methods of in vivo and in vitro production of antigens are further described herein as they relate to pharmaceutical compositions and methods of delivery of combination therapies.

In vitro peptide/polypeptide synthesis

The protein or peptide may be made by any technique known to those skilled in the art including expression of a protein, polypeptide or peptide according to standard molecular biological techniques, isolation of a protein from a natural source, in vitro translation or peptide, or chemical synthesis of a protein or peptide. Nucleotide and protein, polypeptide and peptide sequences corresponding to the various genes have been disclosed and can be referenced by those skilled in the art against well known computerized databases. One database involved is the GenBank and GenPept databases of the national center for Biotechnology information (National Center for Biotechnology Information) on the homepage of the national institute for health (National Institutes of Health) website. The coding region of a known gene may be amplified and/or expressed using techniques disclosed herein, or as known to those of skill in the art. Or various commercially available proteins, polypeptides and peptide preparations are also known to those skilled in the art.

Peptides can be readily synthesized chemically by using reagents that do not contain inclusion bacteria or animal substances (Merrifield RB: "solid phase peptide synthesis I. Tetrapeptides synthesis (Solid phase peptide synthisis I. The synthesis of a tetrapeptide)". J.am. Soc.85:2149-54, 1963). In certain embodiments, the production of antigenic peptides is performed by (1) solid phase synthesis using a multichannel instrument with uniform synthesis and cleavage conditions; (2) purification based on stripping using an RP-HPLC column; and re-washing between peptides, but without exchange; and (3) analysis by a limited set of measurements with highest information value. Regarding a group of peptides for each patient, a standard for manufacturing and quality control (GMP) record (footprint) can be defined, so that only the peptide synthesis of different patients requires a switching sequence step.

Alternatively, the antigen peptide-encoding nucleic acid (e.g., polynucleotide) of the present invention may be used to produce an antigen peptide in vitro. The polynucleotide may be, for example, DNA, cDNA, PNA, CNA, RNA, single-stranded and/or double-stranded, or a naturally occurring or stabilized form of the polynucleotide, for example, a polynucleotide having a phosphorothioate (phosphothiate) backbone, or the like, or a combination thereof, and may or may not include an intron as long as it encodes a peptide. In one embodiment, peptides are made using in vitro translation. There are many exemplary systems (e.g., retic Lysate IVT kit, life Technologies, waltham, MA) that can be used by those skilled in the art.

Expression vectors having the ability to express polypeptides may also be prepared. Expression vectors for various cells are well known in the art and can be selected without undue experimentation. In general, DNA is inserted in an expression vector such as a plasmid in proper orientation and in correct reading frame. If necessary, the DNA may be linked to a nucleotide sequence that is recognized by the desired host (e.g., bacteria) as a suitable transcriptional or translational regulatory control, however such control is typically available to the expression vector. The vector may then be introduced into host bacteria for cloning using standard techniques (see, e.g., sambrook et al (1989) Molecular Cloning, A Laboratory Manual, cold Spring Harbor Laboratory, cold Spring Harbor, n.y.).

Furthermore, expression vectors comprising the isolated nucleotides, as well as host cells comprising the expression vectors are also contemplated. The antigenic peptide may be provided in the form of an RNA or cDNA molecule encoding the desired antigenic peptide. More than 1 antigenic peptide of the invention may be provided by a single expression vector.

In one aspect, the invention provides a method for testing a subject for a disease-related agent, the method comprising the steps of determining the level or amount of follicular T cells reactive with a disease-related agent of the disease in the subject, and comparing the level or amount to a defined baseline.

In one embodiment, the benchmark is a benchmark with respect to at least 1 selected from the group consisting of: disease history, effectiveness of prophylactic or vaccine against disease, effectiveness of therapeutic agent against disease, ability to defend against secondary disease, disease condition, and risk of disease.

In one embodiment, the standard is a standard for the disease of the subject, and the standard may be an average value or median of levels or amounts of follicular T cells reactive with disease-related factors of the disease of a healthy person. When the ratio is higher than the reference, the subject may suffer from a disease.

In one embodiment, the standard is a standard of infection history of the disease in the subject, and the presence or absence of an infectious agent specific immune cell can be used as a standard. When there are specific immune cells of infectious origin, there is a possibility of history of infection.

In one embodiment, the standard is a standard for vaccine effectiveness and reinfection protection against the disease in the subject, and the presence or absence of infection-source-specific immune cells can be used as a standard. When there is specific immune cell of infection source, it can judge that vaccine is effective and has the capability of reinfection.

In one embodiment, the standard is a standard for the effectiveness of a cancer immune drug against the disease in the subject, and the level of immune cells having anti-tumor (epitope) activity can be used as a standard, and the degree of activation or depletion of epitope-specific follicular T cells (tumor-specific follicular T cells) can be used in combination as a standard. When the activity of the epitope specific follicular T cells is increased, the effectiveness of the cancer immune drug can be judged.

In one embodiment, the disease-related factor is an autoimmune disease-related factor, and the determining includes a step of determining a level or amount of follicular T cells reactive with the autoimmune disease-related factor, the criterion being a criterion for a risk or condition of an autoimmune disease associated with an autoantibody in the subject. The presence or absence of immune cells specific for autoimmune disease-related factors can be used as a benchmark. Autoimmune diseases are likely to be encountered when immune cells specific for factors associated with the autoimmune disease are present.

In one aspect, the present invention provides a system for testing a subject for a disease-related substance, the system comprising a reagent or device for determining the level, spatial distribution or amount of follicular T cells reactive with a disease-related factor of the disease in the subject, and a comparing unit for comparing the measurement result obtained by the reagent or device with a predetermined standard.

In one aspect, the present invention provides a method for testing a subject for a disease-related item, the method comprising the steps of providing information about the level, spatial distribution or amount of follicular T cells reactive with a disease-related factor of the disease of the subject, and comparing the information about the level, spatial distribution or amount of follicular T cells with a predetermined reference.

In one aspect, the present invention provides a program for installing a method for testing matters related to a disease of a subject in a computer, the program comprising a program readable by the computer, the program comprising a step of providing information on the level, spatial distribution or amount of follicular T cells reactive with a disease-related factor of the disease of the subject, and a step of comparing the information on the level, spatial distribution or amount of follicular T cells with a predetermined standard.

In one aspect, the present invention provides a computer-readable recording medium storing the above-described program.

< Probe >

In one aspect, the invention provides a polypeptide for presenting a disease-associated factor or a portion thereof, the polypeptide having the ability to elicit follicular T cells specific for the disease-associated factor. Such polypeptides may be used in the examination or diagnosis of the present invention, also referred to as probes.

< probe: variational method

In another aspect, the invention provides an antibody that specifically binds to a TCR that interacts with a disease-related factor or a portion thereof, the antibody having the ability to elicit follicular T cells specific for the disease-related factor. Such antibodies may be used in the examination or diagnosis of the present invention, also referred to as probes.

(treatment and prevention, drug and vaccine)

The present invention may be provided as a medicament. The ingredients contained in the drug or the pharmaceutical composition may be referred to as medicaments and the like. The medicament of the invention may be provided as a vaccine (including a cellular vaccine) or as a probe.

In one aspect, the invention provides a pharmaceutical composition for treating or preventing a disease, the composition comprising a disease-associated factor or a portion thereof having the ability to elicit follicular T cells specific for the disease-associated factor.

In another aspect, the invention provides a vaccine against a disease comprising a disease-associated factor or a portion thereof having the ability to elicit follicular T cells specific for the disease-associated factor.

In another aspect, the invention provides a pharmaceutical composition for treating or preventing a disease, the composition comprising follicular T cells specific for a disease-related factor.

In some aspects, the invention may provide a cellular vaccine against a disease comprising follicular T cells specific for a disease-related factor.

In one embodiment, the disease may be an infection or cancer. In other embodiments, the disease may be a viral infection. In particular embodiments, the compositions of the invention can induce HLA-specific follicular T cells. In some embodiments, the disease-related factor may be an amino acid, a nucleic acid, a virus, or a cell. In other embodiments, the disease-related factor may be a factor that causes the disease directly or indirectly, or may be a factor that causes the disease to develop. For example, where the disease is a viral disease, the disease-related factor may be a virus that causes the viral disease. In some embodiments, the disease-related factor or a portion thereof comprises up to 20 amino acids. In particular embodiments, the follicular T cells of the present invention may be public follicular T cells.

In one aspect, the invention relates to a TCR-T cell therapy.

In some aspects, for example, the invention provides a composition for enhancing immune acquisition against a disease, the composition comprising follicular T cells specific for a disease-related factor.

In one aspect, the invention provides a pharmaceutical composition for treating or preventing a disease, the composition comprising a disease-associated factor or a portion or functional equivalent thereof having the ability to elicit follicular T cells reactive with the disease-associated factor.

In one aspect, the invention provides a vaccine against a disease comprising a disease-associated factor or a portion or functional equivalent thereof having the ability to elicit follicular T cells specific for the disease-associated factor. In one embodiment, the composition or vaccine may induce follicular T cells preferentially, or may induce follicular T cells in addition to follicular T cells.

In one aspect, the invention provides a pharmaceutical composition for treating or preventing a disease, the composition comprising follicular T cells which are reactive to a disease-related factor.

In one aspect, the invention provides a cellular vaccine against a disease comprising follicular T cells reactive to a disease-related factor.

In one embodiment, the follicular T cells of the present invention are reactive with a disease-related factor, at least with a disease-related factor. In other embodiments, in particular embodiments of the infection or cancer, the disease is a viral infection. In another embodiment, the above composition induces HLA-specific follicular T cells. In some embodiments, the follicular T cells are public follicular T cells.

In the present invention, in the case of oral administration, the composition may be formulated into various forms such as tablets, granules, fine granules, powders, capsules, and the like, and may contain additives such as binders, inclusion agents, excipients, lubricants, disintegrants, and wetting agents which are generally used in the preparation. In addition, the preparation for oral administration may be formulated as a liquid such as an aqueous solution for internal use, a suspension, an emulsion, or a syrup, or may be formulated as a dry substance that is redissolved at the time of use.

In the case of parenteral administration, the composition may be prepared in a state of being contained in a unit dose ampoule, a multiprimary container, or a tube, or may contain additives such as a stabilizer, a buffer, a preservative, and an isotonicity agent. The preparation for parenteral administration may be formulated into a powder which is reconstituted with an appropriate carrier (sterilized water or the like) at the time of use.

The "active ingredient" in the present specification means: the composition of the present invention may contain other components in an amount necessary to obtain the desired effect of preventing or suppressing progression of the treatment, as long as the effect is not impaired below a desired level. The drug, composition, and the like of the present invention may be formulated substances. The route of administration of the drug, composition, etc. of the present invention may be any of oral or parenteral route, and may be appropriately set according to the form of the preparation.

Pharmaceutically acceptable salts may be used, such as inorganic acid salts (hydrochloride, hydrobromide, phosphate, sulfate, etc.), or organic acid salts (acetate, propionate, malonate, benzoate, etc.).

Pharmaceutically acceptable salts in the pharmaceutical compositions also include liquids (water, physiological saline, glycerol, and ethanol). Further, an auxiliary agent (wetting agent, emulsifying agent, pH buffering agent, etc.) may be present in the composition. The carrier may be formulated into a tablet, pill, dragee, liquid, gel, syrup, slurry, or suspension form for ingestion by a subject.

The scope of the present invention is a composition present in several modes of administration; the form is not limited to a suitable form for parenteral administration (e.g., injection or infusion (e.g., a large number of instantaneous administrations or continuous infusion)). When the product is injected or infused, it may be administered as a suspension, solution or emulsion in an oily or aqueous medium, and may contain a blending agent (suspending agent, stabilizer and/or dispersing agent). Alternatively, the composition may be dried to recover the composition before use with an appropriate sterilizing liquid.

Once prepared, the compositions of the present invention may be administered directly to a subject. In one embodiment the composition is suitable for administration to a mammal (e.g., a human subject).

The pharmaceutical compositions of the present invention may have any number of routes of administration (including, but not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intraperitoneal, intrathecal, intraventricular (brain), transdermal, topical, subcutaneous, intranasal, enteral, sublingual, intravaginal, or direct routes).

Direct delivery of the composition is typically accomplished via subcutaneous, intraperitoneal, intravenous, intramuscular injection, or to the interstitial space of the tissue. The composition can be administered to a lesion. The dosing regimen may be a single dosing regimen or a multiple dosing regimen. The medication will provide an indication as to the number of administrations (e.g., whether it should be delivered daily, weekly, monthly, etc.). In addition, the number and amount of times and amounts depend on the severity of the symptoms.

The compositions of the present invention can be prepared in a variety of forms. For example, the composition may be prepared as any of a liquid solution or suspension, as an injectable medicament. Can be prepared in a solid form suitable for solution or suspension in a liquid medium prior to injection (freeze-dried compositions (e.g., synagis (registered trademark) and Herceptin (registered trademark) etc.) for reconstitution with sterilized water containing a preservative). The composition may be prepared, for example, as an ointment, cream or powder for topical administration. The composition may be prepared, for example, as a tablet or capsule for oral administration, as a spray, or as a syrup (with any flavor added). The composition may be formulated as a powder or a spray for inhalation, for example, for pulmonary administration. The composition can be made into suppository or pessary. The compositions may be prepared, for example, as drops for nasal, otic or ocular administration. The composition may be in the form of a kit (kit) designed such that the combined composition is reconstituted just prior to administration to a subject. For example, the antibody may be provided in the form of a kit comprising sterile water or sterile buffer.

In another aspect, the invention may provide a vaccine. In addition, the present invention provides a method for preventing diseases and the like using a vaccine and the like in some aspects.

In some embodiments, the pharmaceutical composition of the invention may be a vaccine composition that is administered to humans for the purpose of enhancing immunity. The vaccine composition may comprise one or more adjuvants. Examples of adjuvants contained in the vaccine composition the following examples may be provided in this specification. In some embodiments, the vaccine composition may further comprise a cellular vaccine. In an exemplary embodiment, examples of suitable adjuvants may be listed: (1) Oil-in-water emulsion formulations (with or without other specific immunostimulatory substances such as muramyl polypeptides or bacterial cell wall components), for example MF59 (trademark) (containing 5% squalene, 0.5% tween 80, and 0.5% sorbitol trioleate) and SAF (containing 10% squalene, 0.4% tween 80, 5% pluronic (registered trademark) block polymer L121, and threonyl (thr-) MDP); (2) Saponin auxiliaries such as QS21, STIMULON (trademark) (Cambridge Bioscience, worcester, MA), abisco (registered trademark) (Isconova, sweden), or Iscomatrix (registered trademark) (Commonwealth Serum Laboratories, australia); (3) Complete Freund's Adjuvant (CFA) and Incomplete Freund's Adjuvant (IFA); (4) Oligonucleotides comprising CpG motifs in which the cytosine is unmethylated, i.e.containing at least one CG dinucleotide (e.g. Krieg, vaccine (2000) 19:618-622; krieg, currOpinMolTher (2001) 3:15-24; WO98/40100, WO98/55495, WO98/37919 and WO 98/52581); and (5) a metal salt containing an aluminum salt (alum, aluminum phosphate, aluminum hydroxide, etc.); (6) Saponins and oil-in-water emulsions (e.g. WO 99/11241).

In one aspect, a disease can be treated or prevented by administering to a subject having or at risk of having the disease a pharmaceutical composition comprising a disease-related factor or a portion or functional equivalent thereof that has the ability to elicit follicular T cells reactive with the disease-related factor.

In connection with the treatment or prevention of the present invention, the vaccine is designed in such a way that a substance containing a defined follicular T cell epitope or functional equivalent thereof is combined with (1) other vaccine antigens/nucleic acids for the purpose of B cell induction or the like, or (2) substances for the purpose of immune priming different from B cells+tfh such as cytotoxic T cell epitopes or the like, independently of other antigen expression in viral vectors and VLPs. Vaccine effectiveness the optimization of the combination, number or balance of presented antigens, and the combined adjuvants can be performed by HLA humanized mice. Drug evaluation can be performed to some extent by animals, but it is difficult to fully reproduce human immunity, which is a difficulty when targeting T cells. In the case of tumors, the method of administration is most likely to be local to the tumor, but is not limited thereto.

In one aspect, the treatment or prevention of the present invention may be carried out by performing the steps of:

(1) A step of binding a substance comprising the defined follicular T cell epitope, or a functional equivalent thereof, to other vaccine antigens/nucleic acids, or expressed independently of the viral vector, VLP,

(2) A step of mixing with a substance that elicits an immunity different from B cells or Tfh (of cytotoxic T cell epitopes, etc.), an

(3) A step of administering the substance obtained in (2).

(computer program for evaluating disease of subject or recording medium storing the same, and user device constituting system or part of the system)

In one aspect of the present invention, there is provided a method for evaluating a subject for such a disease, as well as a computer program for implementing such a method or a recording medium storing the same, and a user apparatus constituting a system or a part of the system. Etc

The system 1000 comprises: at least 1 user device 100, a server device 200 connected to at least 1 user device 100 via a network 400, and a database unit 300 (fig. 8) connected to the server device 200.

The user device 100 may be any terminal device such as a smart phone, a tablet computer, a smart watch, a laptop (laptop computer), a desktop computer, or a wearable medical device, in addition to a dedicated medical device. The user device 100 may communicate with the server device 200 through the network 400. Here, the type of the network 400 is not limited. For example, the user device 100 may communicate with the server device 200 via the internet, or may communicate with the server device 200 via a LAN. In fig. 8, 3 user devices 100 are depicted, but the number of user devices 100 is not limited thereto. The amount of the user device 100 may be any number of 1 or more, which may be the same or different.

The server device 200 may communicate with at least 1 user device 100 through the network 400. Further, the server apparatus 200 may communicate with the server portion 300 connected to the server apparatus 200.

For example, the database unit 300 connected to the server apparatus 200 may store information on follicular T cells of a plurality of subjects acquired in advance, any health information on a subject, medical information, and the like. The stored plurality of information may be used, for example, to construct a learning model. For example, the database unit 300 may store the constructed learning model.

Fig. 9 shows an example of the configuration of the user device 100.

The user device 100 includes a communication interface unit 110, an input unit 120, a display unit 130, a storage unit 140, and a processor unit 150.

The communication interface section 110 controls communication via the network 400. The processor unit 150 of the user device 100 may receive information from the outside of the user device 100 through the communication interface unit 110, or may transmit information to the outside of the user device 100. For example, the processor unit 150 of the user device 100 may receive information from the server device 200 through the communication interface unit 110, or may transmit information to the server device 200. The communication interface section 110 may control communication by any method.

The input unit 120 allows a user to input information to the user device 100. Regardless of the manner in which the input unit 120 is configured, the user can input information to the user device 100. For example, when the input unit 120 is a touch screen, a user may input information by touching the touch screen. Alternatively, when the input unit 120 is a mouse, the user may input information by operating the mouse. Alternatively, when the input unit 120 is a keyboard, the user may input information by pressing a key of the keyboard. Alternatively, when the input unit 120 is a microphone, the user may input information by voice.

The display unit 130 may be any display for displaying information.

The storage unit 140 stores a program for executing processing in the user device 100, data necessary for the execution of the program, and the like. The storage unit 140 stores, for example, a part or all of a program for evaluating the following medical techniques: evaluation of history of a subject disease, evaluation of preventive or vaccine effectiveness against a disease, evaluation of effectiveness against a therapeutic agent against a disease, evaluation of ability to defend against a secondary disease, evaluation of disease condition, and evaluation of risk of developing a disease. The storage unit 140 may store application software (application) for installing any function. The storage unit 140 may store application software for obtaining information on the level or amount of follicular T cells by measuring biochemical information on follicular T cells, for example. Thus, the user device 100 has a portion for measuring information on follicular T cells. The storage unit 140 may also store application software for obtaining information on the level or amount of follicular T cells, for example, from biochemical information on follicular T cells. Thus, the user device 100 has a portion that realizes a function of measuring an eye potential. The storage unit 140 may store application software for eyeball information obtained by other devices. Here, regardless of how the program is stored in the storage unit 140. For example, the program may be preloaded in the storage section 140. Alternatively, the program may be downloaded and installed in the storage unit 140 via the network 400. The storage unit 140 may be mounted by any storage device.

The processor unit 150 controls all the operations of the user device 100. The processor section 150 reads the program stored in the storage section 140 and runs the program thereof. This can function as a device for operating the steps desired by the user device 100. The processor unit 150 may be mounted by a single processor or may be mounted by a plurality of processors.

The user device 100 may further include a data acquisition unit 160 in addition to the above configuration. The data acquisition unit 160 is any device for acquiring biochemical information of follicular T cells, which is exemplified by biochemical means such as ELISA. For example, the data acquisition unit 160 is an instrument for realizing ELISA. The data acquisition unit 160 may be, for example, a built-in light absorption device of the user device 100, or may be an external FACS device mounted on the user device 100.

In addition to the above configuration, the user device 100 may further include an additional information acquisition device 170 instead of the data acquisition unit 160, or may further include an additional information acquisition device 170 in addition to the data acquisition unit 160. The additional information acquisition device 170 may be any device for acquiring additional information. The additional information acquisition device 170 is a device that acquires information on, for example, an infectious disease, a vaccine, a cancer, an allergy, and an autoimmune disease. For example, the additional information acquisition device 170 may be a biochemical device such as PCR for identifying an infectious disease in the user device 100, or may be an antigen-antibody reaction measurement device mounted in the user device. For example, when the user device 100 is combined with another medical instrument, the other medical instrument may be used as the additional information acquisition device 170.

In the example shown in fig. 9, each component of the user device 100 is provided in the user device 100, but the present invention is not limited to this. Any of the components of the user device 100 may be provided outside the user device 100. For example, the input unit 120, the display unit 130, the storage unit 140, the processor unit 150, and the image pickup device 160 may be each constituted by a hardware component, and the hardware components may be connected via an arbitrary network. In this case, the type of network is not limited. The hardware components may be connected, for example, by a LAN, by a wireless connection, or by a wired connection. The user device 100 is not limited to a specific hardware configuration. For example, it is within the scope of the present invention that the processor portion 150 may be formed of analog circuitry instead of digital circuitry. The configuration of the user device 100 is not limited to the above-described example as long as the function can be realized.

Fig. 10 shows an example of the structure of the server apparatus 200.

The server device 200 includes a communication interface unit 210, a storage unit 220, and a processor unit 230.

The communication interface section 210 controls communication through the network 400. In addition, the communication interface section 210 also controls communication with the database 300. The processor unit 230 of the server apparatus 200 may receive information from outside the server apparatus 200 through the communication interface unit 210, or may transmit information to outside the server apparatus 200. The processor unit 230 of the server apparatus 200 may receive information from the user apparatus 100 through the communication interface unit 210, or may transmit information to the user apparatus 100. The communication interface section 210 may control communication by any method.

The storage unit 220 stores a program necessary for executing the processing of the server apparatus 200, data necessary for executing the program, and the like. For example, a part or all of a program for analyzing follicular T cells or evaluating a disease therewith is stored. The storage unit 220 stores, for example, application software for evaluating the following medical techniques: application software for obtaining information on the level or amount of follicular T cells, evaluation of histories of a disease in a subject, evaluation of the effectiveness of a prophylactic or vaccine for the disease, evaluation of the effectiveness of a therapeutic agent for the disease, evaluation of the ability to defend against a secondary disease, evaluation of the condition of the disease, and evaluation of the risk of developing the disease. The storage unit 220 may also store application software for acquiring information of follicular T cells or other medical techniques by other means, for example. The storage unit 220 may be mounted by any storage device.

The processor unit 230 controls the overall operation of the server apparatus 200. The processor section 230 reads the program stored in the storage section 220 and runs the program thereof. Thus, the server apparatus 200 can be caused to function as an apparatus that operates desired steps. The processor unit 230 may be mounted on a single processor or may be mounted on a plurality of processors.

In the example shown in fig. 10, the components of the server apparatus 200 are provided in the server apparatus 200, but the present invention is not limited to this. Any of the components of the server apparatus 200 may be provided outside the server apparatus 200. For example, the storage unit 220 and the processor unit 230 may be each constituted by a separate hardware component, and the hardware components may be connected via an arbitrary network. In this case, the type of network is not limited. The hardware components may be connected, for example, by a LAN, by a wireless connection, or by a wired connection. The server apparatus 200 is not limited to a specific hardware configuration. For example, it is within the scope of the present invention that the processor portion 230 may be formed of analog circuitry instead of digital circuitry. The configuration of the server apparatus 200 is not limited to the above example as long as the function can be realized.

In the example shown in fig. 8 to 10, the database 300 is provided outside the server apparatus 200, but the present invention is not limited to this. The database 300 may be provided inside the server apparatus 200. In this case, the database 300 may be mounted on the same storage device as the storage device on which the storage unit 220 is mounted, or may be mounted on a different storage device from the storage device on which the storage unit 220 is mounted. In any case, the database 300 is configured as a storage unit for the server apparatus 200. The configuration of the database 300 is not limited to a specific hardware configuration. For example, the database 300 may be constituted by a single hardware component or a plurality of hardware components. For example, the database 300 may be configured as an external hard disk device of the server apparatus 200, or may be configured as a storage device on a cloud connected via a network.

(general technique)

Molecular biology, biochemistry, microbiology are well known and commonly used in the art for use in this specification, e.g., as described in Current Protocols in Molecular Biology (http:// onlineibrary. Wiley. Com/book/10.1002/0471142727) and Molecular Cloning: ALaboratory Manual (fourier Edition) (http:// www.molecularcloning.com), and the like, which are incorporated by reference in their entirety in relevant parts of this specification.

"or" in this specification is used when "at least 1 or more" of the items listed in the article can be employed. "or" is also the same. When "within" a range of "2 values" is written in this specification, the range also includes the 2 values themselves.

References such as scientific literature, patents, and patent applications cited in this specification are incorporated by reference in their entirety to the same extent as each specifically recited in this specification.

The foregoing description of the preferred embodiments of the present invention has been presented for ease of understanding. The present invention is described below based on examples, but the above description and the following examples are provided for illustrative purposes only and are not provided for limiting the invention. Therefore, the scope of the present invention is not limited to the embodiments or examples specifically described in the present specification, but is limited only by the scope of the claims.

[ example ]

In this example, the following experiment was performed based on the declaration of helsinki, with the necessary informed consent (formed consent) from the subject, following the ethical regulations prescribed in university. The reagents used may be those obtained from the pharmaceutical industry, sigma-Aldrich, etc., in addition to those described in the examples.

Example 1 viral case (in the case of general case)

In this example, a subset of SARS-CoV-2 specific T cells and their clonotypes were analyzed using a single cell based RNA sequence platform (Chromium, 10 xGenomics).

(isolation and plasma collection of Peripheral Blood Mononuclear Cells (PBMC))

Peripheral Blood Mononuclear Cells (PBMCs) were prepared from 6 persons including healthy donors and virus infected patients (e.g., influenza virus infected patients). The preparation was carried out as follows. Whole blood was collected in heparin-coated tubes and centrifuged at 1500rpm for 10 minutes to separate cells from plasma. Plasma was removed from the cell pellet and stored at-80 ℃. PBMCs were then isolated by density gradient sedimentation and erythrocytes were lysed using ACK lysis buffer. The isolated PBMC were cryopreserved at-80℃by STEM-CELLBANKER (Zenoaq Resource).

Cryopreserved PBMCs were thawed and washed with RPMI1640 medium supplemented with 5% human AB serum. Stimulation of 5X 10 by inactivated virus (e.g.influenza virus) containing 1. Mu.g/ml of S protein, recombinant S protein (1. Mu.g/ml), pool of S peptides (1. Mu.g/ml of each peptide) or pool of M+N peptides (1. Mu.g/ml) ⁵ PBMC (stimulatory proteins are selected appropriately according to the virus recombinant viral proteins are prepared according to well known literature). The PepMix influenza virus (various proteins) and the like are commercially available. (e.g., funakoshi company). Then, stained with anti-human CD3 (HIT 3 a), CD69 (FN 50), CD137 (4B 4-1) and TotalSe (trademark) -C hash tags (all available from BioLegend Inc.) for 20 hours at 37 ℃. CD3+CD69+ or CD3+CD137+ cells were sorted by cell sorter (cell sorter) SH-800S cells (SONEY) and analyzed for RNA expression and TCR sequences using single cell VDJ-RNA-seq analysis methods as described below.

(Single cell based transcript and TCR repertoire analysis)

Single cell capture and library preparation were performed using the following reagents. Chromium SingleCell 5'Library&Gel head Kit, PN-1000165; chromium Next GEM Chip G Single Cell Kit, PN-1000120; chromium Single Index Kit T Set A, PN-1000213; chromium Single Cell 5'Feature Barcode Library Kit, PN-1000080; single Index Kit N Set A, PN-1000212; chromium single cell V (D) J concentration kit, human T cells, PN-1000005. Loading of microfluidic chip at Chromium comprises about 2×10 ⁴ Single cell suspensions of single cells, gel bead latex droplets (Gel Bead in Emulsion) of single cells were generated using a chromasum controller (10 XGenomics) as directed by the manufacturer. RNA from the barcoded cells of each sample was then reverse transcribed in gel bead latex droplets using Veriti Thermal Cycler (Thermo Fisher Scientific), following the experimental procedure of the manufacturer, all subsequent steps for generating single cell libraries were performed in 14 cycles for cDNA amplification. Next, about 50ng of cDNA was used for amplification of the 14 cycles gene expression library while the cDNA of the TCR library was concentrated and the library was constructed. Fragment size of the library was confirmed by Agilent 2100Bioanalyzer (Agilent). The library was sequenced as a double ended model (read 1:28bp; read2:91 bp) using an Illumina NovaSeq 6000. Raw data (raw read) were processed through Cell Ranger3.1.0 (10 XGenomics). Gene expression-based clustering (v 3.1, hafemeister, C., satija, R.normalization and variance stabilization of single-cell RNA-seq data using regularized negative binomial regression. Genome Biol 20,296 (2019): https:// doi.org/10.1186/s 13059-019-1874-1) was performed using the Seurat R package. Briefly, for cells with mitochondrial content exceeding 10%, cells with genes detected below 200 or above 4000 were regarded as deviant (dying cells, empty droplets, dimers, respectively) and removed by filtration. Since normalization uses the Seurat SC Transform function, all samples are processed simultaneously, the data is merged without performing batch effect correction. HashTag oligomerization inverse multiplexing was performed with HashTag umi counts normalized using CLR, using Python script for clonotypes matched to gene expression data by droplet bar code. Only remain allocated a single hash tag and beta- Cells of the chain clonotype were used for downstream analysis.

(bulk TCR sequencing) (bulk TCR-seq)

1 to 3X 10 of the solution was dissolved by QIAzol ⁵ The full-length cDNA was then synthesized using SMARTer Technology (TakaraBio) and the variable regions of the TCR alpha and beta genes were amplified using TRAC/TRBC specific primers. After sequencing the variable region amplicons, clonotypes (TR (A/B) V and TR (A/B) J genes and Complementarity Determining Region (CDR) 3) were assigned to each pairing read using MiXCR software (Bolotin, D., poslafsky, S., mitrophanov, I.et al. MiXCR: software for comprehensive adaptive immunity profiling. Nat Methods 12,380-381 (2015): https:// doi.org/10.1038/nmeth.3364). For each α/β clonotype, the ratio of reads for its clone was divided by the total number of reads for the α/β strand to define the amplification, and the ratio was converted to log10 for mapping and statistical analysis.

From the UMAP map and single cell gene expression, it was confirmed that circulating Tfh clusters consisted of cells expressing Tfh-related genes of CD200, PDCD1, ICOS, CXCL13, CD40LG, etc. Further, more than 1100 pairs of TCRs are specified in the Tfh cluster.

Wherein a TCR αβ pair shared between patients is detected.

These tcrαβ pairs were designated as clones 1 and 2 from patients X1 and X2, which shared the same use of vα, jα, vβ and jβ, respectively. Furthermore, it was found that the CDR 3. Alpha. Sequences of clones 1 and 2 were identical and that CDR 3. Beta. Was identical except for one amino acid.

In patient X2, clone 2 was detected as 2 different barcode cells sharing the exact same TCR sequence.

To examine the antigen specificity of TCR, clonotypes 1 and 2, TCR α and β chains were transfected into TCR-deleted T cell hybridomas, respectively, to construct TCR recombinant cd3+ cells.

These TCR transfectants were stimulated with various antigens in the presence of epstein-barr virus (EBV) transformed B cells from the corresponding individual patients.

The responsiveness of clones 1 and 2 was confirmed. This was confirmed to be consistent with the initial antigen specificity revealed in the single cell analysis.

Since activation in the absence of antigen presenting cells is precluded, activation is confirmed to be mediated by MHC molecules.

Of the 2 pools that were pooled, only the latter pool stimulated clones 1 and 2, and thus it was known that the antigen peptide was contained at a specific position in the latter.

However, it was confirmed that otherwise available peptide pools containing a number of peptides did not activate clones 1 and 2.

From these results, the positions of the epitopes of clones 1 and 2 can be confirmed.

Incidentally, in order to further reduce the number of epitope candidate peptides, HLA alleles of restriction clones 1 and 2 were determined.

Antigen Presenting Cells (APCs) from both patients X1 and X2 were found to activate clones 1 and 2. This shows that APCs can be interchanged.

Thus, DRB1 x 15: 01. DPA1 x 02:02-DPB1 x 05: 01. DQA1 x 01: MHC class II alleles shared between X1 and X2 such as 02-DQB 1X 06 are each candidates for HLA. Then, to examine the recognition of the S peptide by clones 1 and 2, these alleles were transfected separately.

Both clones 1 and 2 were found to be DRA-only 01:01 and DRB x 15:01 in response to S peptide pool #2 in the presence, since DRA is monomorphic, the postulated response is determined by DRB x 15: 01.

Here, using NetMHC server software (http:// www.cbs.dtu.dk/services/NetMHC ipian /), the search prediction can be compared with DRA-DRB1 x 15:01 bound candidate peptide.

Some strong binders are predicted to be candidates within the reduced area of a particular viral protein. Thus, by synthesizing 2 peptides to stimulate clones 1 and 2, a particular peptide can be determined as an epitope for both clones 1 and 2.

It was found that the TCRs of clones 1 and 2 differ in one amino acid in CDR 3. In this case, these 2 TCRs can be interpreted as suggesting that clones 1 and 2 are public clones, since they recognize the same epitope presented by the same MHC molecule and originate from different donors. This epitope may be interpreted as having a low identity with the corresponding amino acid sequences of other influenza viruses.

In fact, in expression DRA-DRB1 x 15: in the presence of APC of 01, both clones 1 and 2 were completely unresponsive to peptide pools and whole proteins from other viral proteins. Furthermore, this suggests that clone 1/2 has a low probability of cross-reacting with the relevant influenza virus.

Next, examine division DRB1 x 15: whether or not an MHC class II allele other than 01 can present the antigenic peptide recognized by clone 1/2.

As a result, specific amino acid regions can also be predicted as for DRB1 x 15:02 and 15:01. Furthermore, DRB1 x 15:01 and 15: the amino acid sequence identity between 02 is 99.6%. Therefore, DRB1 x 15 is presumed: 02 is also an allele responsible for epitope recognition, and APC from another person can be tested, with DRB1 x 15:02 but without DRB1 x 15:01. as a result, DRB1 x 15 was determined in the presence of this specific peptide: 02 are able to stimulate both clones 1 and 2.

Considering DRB1 x 15 in japanese population: 02 (10.6%) and 15:01 (7.7%) frequency (http:// hla.or.jp /), clones 1 and 2 were expected to respond to the peptide in 18.3% of the population if these clonotypes were present. The same restriction specificity can be checked for other HLA types.

Next, the occurrence of clonotypes of clone 2/3 in the population considered to be free of influenza virus was examined, and it was found that there was a certain degree of clonotype of TCR.beta.of clone 1/2 in the population. This is the same ratio as in the present embodiment.

Secondly, considering that clone 1/2 is the virus-specific T cell showing Tfh profile, to promote defensive humoral immunity, clone 1/2 is assumed to clonally proliferate at the time of infection, as examined below.

As DRB1 x 15:01/15:02 the frequency of clonotypes of clone 1/2 of healthy individuals and individuals recovered from infection was investigated in public databases of citizens in the United states (immune response activity of the COVID-19 event; immuneRace, https:// ImmuneRace.adaptive biotech.com /) which are not alleles of the small population worldwide (cf. Http:// www.allelefrequencies.net/tools/report. Aspx). Clonotypes were detected with some frequency in the healthy and recovery groups, respectively. In particular, since a significant increase in this clonotype frequency between each individual was observed in the recovery group compared to healthy controls, this suggests that specific protein-specific clones were clonally amplified in different individuals at the time of infection. Interestingly, clonotypes of clone 1/2 occur more frequently in recovery patients than DRB1 x 15 in the world: 01 or 15:02 allele frequency (11.3%), it is therefore believed that clone 1/2 may recognize other such virus-derived epitopes presented by other HLA alleles.

In summary, the initial specificity of public T cell clones that recognize SARS-CoV-2 and its epitopes are believed to be helpful in the development of diagnostic strategies or options and next generation vaccines.

Example 2 viral case (SARS-CoV-2 case)

In this example, a single cell-based RNA sequence platform (Chromium, 10xGenomics Co.) was used to analyze the SARS-CoV-2 specific T cell subpopulation and its clonotypes.

(isolation and plasma harvesting of Peripheral Blood Mononuclear Cells (PBMC))

Peripheral Blood Mononuclear Cells (PBMC) were prepared from 6 persons including healthy donors and patients with convalescence from COVID-19. The preparation was carried out as follows. Whole blood was collected in heparin-coated tubes and centrifuged at 1500rpm for 10 minutes to separate cells from plasma. Plasma was removed from the cell pellet and stored at-80 ℃. PBMCs were then isolated by density gradient sedimentation and erythrocytes were lysed using ACK lysis buffer. The isolated PBMC were stored by STEM-CELLBANKER (Zenoaq Resource) at-80 ℃.

Table 1: characterization of participants

[ Table 1 ]

Table 2: classification of severe degree of covd-19 disease in japan

[ Table 2 ]

Cryopreserved PBMCs were thawed and washed with RPMI1640 medium supplemented with 5% human AB serum. SARS-CoV-2 stimulation was by inactivation with 1. Mu.g/ml of S protein, recombinant S protein (1. Mu.g/ml), pool of S peptides (1. Mu.g/ml of each peptide) or pool of M+N peptides (1. Mu.g/ml). (inactivated SARS-CoV-2 virus was supplied by Minn and Zhongshan (institute of microbiological diseases of Osaka university) recombinant SARS-CoV-2 protein was formulated as described by Amanat F, et al bioRxiv 2020. Pepmix SARS-CoV-2 (spike glycoprotein) (including pools #1 and # 2) was purchased from JPT Peptide Technologies. Peptivator SARS-CoV-2prot_M and N was purchased from MiltenyiBiotec.) then stained with anti-human CD3 (HIT 3 a), CD69 (FN 50), CD137 (4B 4-1) and TotalSe (trademark) -C hash tags (all from BioLegend Corp.) at 37℃for 20 hours. The CD3+CD69+ or CD3+CD137+ cells were sorted by cell sorter SH-800S cells (SONY) and analyzed for RNA expression and TCR sequences using single cell VDJ-RNA-seq analysis methods as described below.

(Single cell based transcript and TCR repertoire analysis)

Single cell capture and library preparation were performed using the following reagents. Chromium Single Cell 5' library&Gel head Kit, PN-1000165; chromium Next GEM Chip G Single Cell Kit, PN-1000120; chromium Single Index Kit T Set A, PN-1000213; chromium Single Cell 5'Feature Barcode Library Kit, PN-1000080; single Index Kit N Set A, PN-1000212; chromium single cell V (D) J concentration kit, human T cells, PN-1000005. Loading of microfluidic chip at Chromium comprises about 2×10 ⁴ Single cell suspensions of single cells, gel bead latex droplets (Gel Bead in Emulsion) of single cells were generated using a chromasum controller (10 XGenomics) as directed by the manufacturer. RNA from the barcoded cells of each sample was then reverse transcribed in gel bead latex droplets using Veriti Thermal Cycler (Thermo Fisher Scientific), following the manufacturer's experimental procedure (protocol), all subsequent steps for generating single cell libraries were performed in 14 cycles for cDNA amplification. Next, cDNA concentration in TCR library and textWhile the library was being constructed, approximately 50ng of cDNA was used for 14 cycles of gene expression library amplification. Fragment size of the library was confirmed by Agilent 2100Bioanalyzer (Agilent). The library was sequenced as a double ended model (read 1:28bp; read2:91 bp) using an Illumina NovaSeq 6000. Raw data were processed through Cell Ranger3.1.0 (10 XGenomics). Gene expression-based clustering (v 3.1, hafemeister, C., satija, R.normalization and variance stabilization of single-cell RNA-seq data using regularized negative binomial regression. Genome Biol 20,296 (2019): https:// doi.org/10.1186/s 13059-019-1874-1) was performed using the Seurat R package. Briefly, for cells with mitochondrial content exceeding 10%, cells with genes detected below 200 or above 4000 were regarded as deviant (dying cells, empty droplets, dimers, respectively) and removed by filtration. Since normalization uses the Seurat SC Transform function, all samples are processed simultaneously, the data is merged without performing batch effect correction. HashTag oligomerization inverse multiplexing was performed with HashTag umi counts normalized using CLR, using Python script for clonotypes matched to gene expression data by droplet bar code. Only cells assigned a single hash tag and β -strand clonotype were retained for downstream analysis.

(batch TCR sequencing)

1 to 3X 10 of the solution was dissolved by QIAzol ⁵ The full-length cDNA was then synthesized using SMARTer Technology (TakaraBio) and the variable regions of the TCR alpha and beta genes were amplified using TRAC/TRBC specific primers. After sequencing the variable region amplicons, clonotypes (defined as TR (A/B) V and TR (A/B) J genes and Complementarity Determining Region (CDR) 3) were assigned to each pairing read using MiXCR software (Bolotin, D., poslafsky, S., mitrophanov, I.et al. MiXCR: software for comprehensive adaptive immunity profiling. Nat Methods 12,380-381 (2015), https:// doi.org/10.1038/nmeth.3364). For each α/β clonotype, the ratio of reads for its clone was divided by the total number of reads for the α/β strand to define the amplification, and the ratio was converted to log10 for mapping and statistical analysis.

From the UMAP map and single cell gene expression, it was confirmed that circulating Tfh clusters consisted of cells expressing Tfh-related genes of CD200, PDCD1, ICOS, CXCL13, CD40LG, etc. (fig. 1A). Further, 120 pairs of TCRs were specified in the Tfh cluster (table 3).

[ Table 3-1 ]

[ Table 3-2 ]

[ Table 3-3 ]

[ tables 3-4 ]

[ tables 3-5 ]

[ tables 3-6 ]

[ tables 3-7 ]

[ tables 3-8 ]

[ tables 3-9 ]

[ tables 3 to 10 ]

[ tables 3-11 ]

Among these, TCR αβ pairs shared between patients were detected.

These tcrαβ pairs were designated as clones 1 and 2 from patients X1 and X2, which shared the same use of vα, jα, vβ and jβ, respectively. Furthermore, it was found that the CDR3 α sequences of clones 1 and 2 were identical, and the CDR3 β was identical except for one amino acid (table 4 and fig. 2). Clones 1 and 2, although from different subjects, were presumed to recognize the same epitope from sequence similarity. In addition, the epitope is determined from Tfh clustering, and is expected to be an epitope that is easy to induce Tfh.

[ Table 4 ]

For patient Ts-018, clone 2 was tested as two different bar code cells sharing identical TCR sequences. To examine the antigen specificity of TCR, clonotypes 1 and 2, TCR α and β chains were transfected into TCR-deleted T cell hybridomas, respectively, TCR recombinant cd3+ cells were constructed (fig. 1B).

Clones 1 and 2, although responsive to recombinant S protein and S peptide pool, did not respond to m+n peptide pool. This is consistent with the initial antigen specificity revealed in single cell analysis (fig. 3A).

Since activation in the absence of antigen presenting cells was excluded, activation was confirmed to be mediated by MHC molecules (fig. 3B).

Of the 2 pooled vials (visual) #1 (S1-643) and #2 (S633-1273), only the latter pool stimulated clones 1 and 2, and thus it was seen that the antigenic peptides were contained in the S proteins 633-1273 (FIG. 3C).

However, the otherwise available SARS-CoV-2S peptide pool (Peptivator (registered trademark)) containing 175 peptides corresponding to positions 304-338, 421-475, 492-519, 683-707, 741-770, 785-802 and 885-1273 (FIG. 4) did not activate clones 1 and 2 (FIG. 3D).

These results suggest that the epitopes of clone 1 and clone 2 are located in amino acids 633-682, 708-740, 771-784 or 803-884 of the S protein (FIG. 4).

Then, to further reduce the number of epitope candidate peptides, HLA alleles of restriction clones 1 and 2 were determined.

Antigen Presenting Cells (APCs) from both patient Ts-017 and Ts-018 were found to activate clones 1 and 2. This shows that APCs can be interchanged (fig. 3E).

Thus, DRB1 x 15: 01. DPA1 x 02:02-DPB1 x 05: 01. DQA1 x 01: MHC class II alleles shared between Ts-017 and Ts-018, such as 02-DQB 1. Times.06, are candidates for HLA, respectively. Then, to examine the recognition of the S peptide by clones 1 and 2, these alleles were transfected separately.

Both clones 1 and 2 were found to be DRA-only 01:01 and DRB x 15:01 in the presence of response S peptide pool #2, since DRA is monomorphic, it is shown that responsiveness is represented by DRB x 15:01 (fig. 5B).

Thus, using NetMHC server software (http:// www.cbs.dtu.dk/services/NetMHCITman /), the search was expected to be with DRA-DRB 1. Times.15: 01 (fig. 5C).

Predicting some strong adhesives (S _828-846 S and S _864-882 ) Is a candidate within the reduced region of the S protein (fig. 5D). Thus, by synthesizing 2 peptidesStimulating clones 1 and 2, peptide S _864-882 The epitopes determined as those of clones 1 and 2, 2 (fig. 5E).

Although the TCRs of clone 1 and 2 are different in one amino acid in CDR3 (Pro and Gln at position 89), clone 1 and 2 are suggested to be public clones since both TCRs recognize the same epitope presented by the same MHC molecule and originate from different donors. The epitope has low identity with the corresponding amino acid sequences of other influenza viruses.

In fact, in expression DRA-DRB1 x 15:01, clones 1 and 2 were both completely unresponsive to peptide pools and whole proteins from the HCoV-OC43S protein (FIG. 6A). Furthermore, since none of the other hcos, including severe acute respiratory syndrome coronavirus (SARS-CoV) and middle east respiratory syndrome related coronavirus (MERS-CoV), have the same epitope (fig. 6B), it is shown that clone 1/2 has a low probability of cross-reacting with the related human coronavirus.

Next, division DRB1 x 15 was investigated: whether or not an MHC class II allele other than 01 can present the antigenic peptide recognized by clone 1/2.

As a result, S can also be _864-882 Predicted as for DRB1 x 15:02 and 15:01 (fig. 6C). Furthermore, DRB1 x 15:01 and 15: the amino acid sequence identity between 02 is 99.6%. Therefore, DRB1 x 15 is presumed: 02 is also an allele responsible for epitope recognition and an APC from another person was tested, with DRB1 x 15:02 but without DRB1 x 15:01. as a result, it can be seen that S _864-882 DRB1 x 15:02 are able to stimulate both clones 1 and 2 (fig. 6D).

Consider DRB1 x 15 in the japanese population: 02 (10.6%) and 15:01 (7.7%) frequency (http:// hla.or.jp /), clones 1 and 2 were expected to respond to the peptide in 18.3% of the population if these clonotypes were present. In this example, clone 1/2 was confirmed among 2 of 6 persons.

Next, the occurrence of clonotypes of clone 2/3 in Japanese donor samples before SARS-CoV-2 pandemic was examined, and as a result, it was found that 9 out of 27 persons did not have a TCR.beta.clonotype of clone 1/2. This is the same ratio as in the present embodiment.

Secondly, considering clone 1/2 is a SARS-CoV-2 specific T cell that shows a Tfh profile (profile), in order to promote defensive humoral immunity, it is assumed that clone 1/2 clonally proliferates at the time of infection, and the following investigation was performed.

As DRB1 x 15:01/15:02 the frequency of clonotypes of clone 1/2 in healthy individuals (n=786) and individuals recovering from a covd-19 infection (n=1, 413) were investigated in the world's alleles that are not the minor (see http:// www.allelefrequencies.net/tools/report. Aspx), the public database of citizens in the united states (immune response activity for the covd-19 event; immunerate, https:// ImmuneRace. Adaptive biotech. Com /). Clonotypes were detected in the healthy and recovery groups at 17% and 24%, respectively (fig. 6E). In particular, since a significant increase in the frequency of this clonotype between individuals was observed in the covd-19 recovery group compared to healthy controls (p=0.0028), this suggests that this S protein-specific clone was clonally amplified in different individuals at the time of infection (fig. 4E below). Interestingly, clone type 1/2 occurred more frequently in recovery patients than DRB1 x 15 in the world: 01 or 15:02 allele frequency (11.3%), it is therefore believed that clone 1/2 may recognize epitopes from other SARS-CoV-2 that are presented by other HLA alleles.

In summary, the initial specificity of public T cell clones that recognize SARS-CoV-2 and its epitopes is believed to be helpful in the development of diagnostic strategies or options and next generation vaccines.

Example 3

Restimulation assay of Peripheral Blood Mononuclear Cells (PBMC) of peptides by example 1

PBMC 1.0X10 s obtained from the experimental participants of example 1 ⁵ cells were suspended in 100. Mu.L of RPMI1640 medium (supplemented with 5% human serum, penicillin/streptomycin) and the peptide of example 1 was added to 1. Mu.g/ml and incubated in 96-well plates (U bottom) (Day 0).

IL-2 was then added to Day4 and Day7 to a concentration of 10IU/ml. Medium passaging (passage) was performed appropriately.

Cells were sorted at Day10 using a cell sorter (cell sorter) SH-800S (SONY) as follows. Sorting was performed in the order FSC SSC ligation, PI negative gateing, CD3positive ligation, and CD4positive cells using CD3, CD4 antibodies, and PI staining. At this time, FCM analysis was also performed using CXCR5, CD45RA, PD-1 antibody staining.

Sorting the obtained CD4positive cells 2.0X10 ⁴ cells and immortalized B cells from patients 3.0X10 by EBV infection ³ cells were suspended in 50. Mu.L of RPMI1640 medium (5% human serum) and a plurality of wells (wells) were cultured in a 96-well plate (U bottom). SARS-CoV-2S is added to half of them _864-882 The peptide was set at 1. Mu.g/ml.

After 24 hours, the supernatant of the medium was recovered and assayed for human IL-21 using ELISA.

As a result of the measurement, it was found that the concentration of IL-21 in the medium was higher when stimulated with the peptide of example 1 than when not stimulated. From this result, it was revealed that the follicular T cells promote the formation of a hair center and the production of an anti-affinity antibody by stimulation with the peptide of example 1.

Example 4

By SARS-CoV-2S _864-882 Restimulation assay of Peripheral Blood Mononuclear Cells (PBMC)

PBMC obtained from the experimental participants were 1.0X10 ⁵ cells were suspended in 100. Mu.L of RPMI1640 medium (supplemented with 5% human serum, penicillin/streptomycin) and SARS-CoV-2S was added _864-882 The peptide was incubated in 96-well plates (U bottom) to 1. Mu.g/ml (Day 0).

IL-2 was then added to Day4 and Day7 to a volume of 10IU/ml. Passaging of the medium was performed appropriately.

Sorting the obtained CD4positive cells 2.0×10 ⁴ cells and immortalized B cells from patients 3.0X10 by EBV infection ³ cells were suspended in 50. Mu.L of RPMI1640 medium (5% human serum) and cultured in a plurality of wells of a 96-well plate (U bottom). SARS-CoV-2S is added to half of them _864-882 The peptide was brought to 1. Mu.g/ml.

After 24 hours, the culture supernatant was recovered and assayed for human IL-21 by ELISA. The measurement results are shown in FIG. 7.

As a result of the measurement, it was found that SARS-CoV-2S was used _864-882 In the case of peptide stimulation, the IL-21 concentration in the medium was high compared to the case of no stimulation. From the results, it was revealed that SARS-CoV-2S was used _864-882 Peptide stimulation, the formation of hair-generating centers by follicular T cells, and the production of anti-affinity antibodies are promoted.

Example 5 evaluation of infection histories

In this example, the infection history was evaluated.

Although evaluation of infection history is often performed based on antibodies, it is advantageous to determine immune cells specific to the origin of infection in terms of investigation of infection history because specific immune cells are maintained longer than antibodies. The T cell assay using a probe containing an epitope specific to the infectious agent is useful as a method for easily detecting immune cells established and maintained after infection. The presence or absence of T cells that react with the probes can be readily detected by ELISPOT using Peripheral Blood Mononuclear Cells (PBMCs) or by a system using a flow cytometer. If a probe-reactive T cell is detected, it is determined that there has been a past infection.

(materials and methods)

(Material)

In the case of influenza virus

Influenza A, hemagglutin, brisbane (funakoshi) was used as antigen.

In the case of enterohemorrhagic Escherichia coli infection

Lipopolysaccharide (Wako) from Escherichia coli O157 was used as an antigen.

In the case of fungal pneumonia

A standard strain (KWIK-STIK 2 pack) from Aspergillus fumigatus of ATCC 204305 01021P (AS ONE) was used AS antigen.

(Experimental procedure)

The presence or absence of infection-source-specific immune cells in the peripheral blood of a subject is examined by an ELISPOT test. In the ELISPOT test used herein, plates coated with antibodies that detect IL-21 produced by follicular T cells were used. Peripheral Blood Mononuclear Cells (PBMCs) were added to the wells and stimulated with the antigen for 36 hours. The secreted IL-21 binds to the capture antibody at the periphery of the producer cell. After removing cells by washing, a cytokine antibody for detection was added, and spots were detected. As a negative control, the measurement was performed without stimulation with an antigen from an infectious source.

(results)

When IL-21 secretion was confirmed, it was confirmed that there was a history of infection.

( Example 6: evaluation of vaccine effectiveness and evaluation of Reinfection defense ability )

In this example, vaccine effectiveness evaluation and reinfection defense capacity evaluation were performed.

Since specific immune cells are maintained longer than antibodies and respond rapidly upon reinfection, immune cells can be evaluated for their defenses. In addition, since follicular T cells are important for induction and enhancement of humoral immunity (affinity maturation) in the fractionation of a plurality of T cells, it can be determined that a subject having T cells that react with a probe containing an epitope of the infection origin that induces follicular T cells has a stronger or more superior infection defense ability. Therefore, it is considered to be effective to examine the presence or absence of probe-reactive cells in PBMC as an immunoassay established after infection or after vaccine intake. The evaluation method may be performed by using ELISPOT or flow cytometry in the same manner as described above. These methods can alter the cytokine to be detected, or can detect whether or not the T cell that reacts with the epitope probe is actually a follicular T cell by combining the surface antigen probes, or to what extent the ratio is, and can design an experimental system based on the detection sensitivity or the judgment standard.

(materials and methods)

(Material)

In the case of influenza virus

Influenza A, hemagglutin, brisbane (funakoshi) was used as antigen.

In the case of enterohemorrhagic Escherichia coli infection

Lipopolysaccharide (Wako) from E.coli 0157 was used as antigen.

In the case of fungal pneumonia

(Experimental procedure)

(results)

When IL-21 secretion was confirmed, it was confirmed that the defenses of immune cells were effective.

( Example 7: evaluation of cancer immune drug (immune checkpoint etc.) effectiveness )

In this example, the effectiveness of cancer immune drugs (immune checkpoints, etc.) was evaluated.

If a humoral immune response against a cancer antigen is elicited, a higher antitumor property can be expected. Therefore, it is considered that a test body in which follicular T cells inducing an antitumor humoral immune response are detected more, the antitumor immunity is easily activated, and an antitumor immunity-enhancing agent such as an immune checkpoint inhibitor is more effective.

It is considered that evaluating the reactivity of T cells obtained from a tumor biopsy sample or peripheral blood with an epitope probe, evaluating the activity of anti-tumor immunity of a subject contributes to the selection of a therapeutic method. Furthermore, by combining an epitope probe and a flow cytometer, it is expected to examine whether or not the activity of anti-tumor immunity is maintained by evaluating the activation or depletion degree of epitope-specific follicular T cells (tumor-specific T cells) at the time of administration. This is effective in determining whether or not an immune checkpoint inhibitor is effective in the future, such as whether or not the treatment should be discontinued and other treatment methods should be searched for.

(materials and methods)

(Material)

In the case of lung cancer

As antigen, lung cancer marker CEA was used.

In the case of gastric cancer

As antigen, CA19-9, a gastric cancer marker, was used.

(Experimental procedure)

The presence or absence of antigen-specific immune cells in the peripheral blood of a subject is examined by an ELISPOT assay. In the ELISPOT assay used herein, a plate coated with antibodies to IL-21 produced by follicular T cells at the detectable site is used. Peripheral Blood Mononuclear Cells (PBMCs) were added to the wells and stimulated with the antigen for 36 hours. The secreted IL-21 binds to the capture antibody at the periphery of the producer cell. After removing cells by washing, a cytokine antibody for detection was added, and spots were detected. As a negative control, the assay was performed without antigen-based stimulation.

(results)

When an increase in IL-21 secretion was confirmed as compared with the negative control, it was confirmed that the cancer immune drug effectively acted.

( Example 8: risk assessment (when not suffering from an autoimmune disease) and disease assessment associated with autoantibodies )

In this example, the risk of autoimmune disease (when not diseased) accompanied by autoantibodies and the disease condition were evaluated.

There are many autoimmune diseases accompanied by autoantibodies, but among them, the presence or absence or increase or decrease of follicular T cells interacting with B cells producing autoantibodies in diseases considered to be pathogenic by autoantibodies (e.g., ANCA-related vasculitis) are considered to be associated with onset of maturation of B cells and exacerbation/recurrence of the disease. Thus, it is thought that by detecting follicular T cells presenting epitopes from disease-specific autoantigens, disease risk (pre-onset) or disease activity can be assessed.

As a test method, probe-reactive cells of PBMCs sampled from a subject are examined using ELISPOT or flow cytometer.

(materials and methods)

(Material)

In the case of allergic dermatitis

Autoantibodies causing allergic dermatitis are used as antigens.

In the case of allergic rhinitis

Autoantibodies causing allergic rhinitis are used as antigens.

Asthma condition

The antigen uses autoantibodies that cause asthma.

(Experimental procedure)

The presence or absence of antigen-specific immune cells in the peripheral blood of a subject is examined by an ELISPOT assay. In the ELISPOT test used herein, a plate coated with an antibody that detects IL-21 produced by follicular T cells was used. Peripheral Blood Mononuclear Cells (PBMCs) were added to the wells and stimulated with the antigen for 36 hours. The secreted IL-21 binds to the capture antibody at the periphery of the producer cell. After removing cells by washing, a cytokine antibody for detection was added, and spots were detected. As a negative control, the assay was performed without antigen-based stimulation.

(results)

When an increase in IL-21 secretion was confirmed as compared with the negative control, it was confirmed that the risk of cancer disease (before onset) or the disease activity was high.

Example 9 digital health application example

As described in the examples above, epitope-specific T cell responses may be applicable to a variety of diseases. On the one hand, the presence of diseases where epitopes or antigens themselves are unclear is also a fact, and thus data of follicular T cell responses to various epitopes including known disease-related epitopes of various patients are shared during the study, and are used as large data for obtaining newly discovered basal discs. As a test method, examination of probe-reactive cells of PBMCs sampled from a subject using ELISPOT or flow cytometer is considered.

(materials and methods)

(Material)

Systemic lupus erythematosus with unclear antigens is considered as a subject disease.

In contrast, epitopes derived from antigens such as known infectious diseases or randomly prepared epitope panels (panels) were used to evaluate the responses or levels of follicular T cells to the respective epitopes.

(Experimental procedure)

(results)

The negative control and the IL-21 secretion amount information and clinical information of each epitope are shared and stored as data, and are used for future development of diagnostic methods, disease differentiation, and treatment method selection methods.

The present invention is not limited to the above-described embodiments, and various modifications can be made within the scope of the present invention.

(remark)

As described above, although the present invention is exemplified using the preferred embodiments thereof, the present invention should not be construed as being limited to these embodiments. It is intended that the scope of the invention be construed solely by the appended claims. It is to be understood that those skilled in the art can practice the present invention within the equivalent scope from the description of the specific preferred embodiments of the present invention based on the description and technical knowledge of the present invention. It will be understood that the patents, patent applications, and literature cited in this specification are to be incorporated by reference as if specifically set forth in this specification. The present application claims the preference of japanese patent application publication No. 2021-42670, filed on 3/16 of 2021, the contents of which are incorporated herein by reference in their entirety.

[ INDUSTRIAL APPLICABILITY ]

The invention can be applied in the fields of vaccine development, cell therapy, diagnosis technology and the like.

Claims

1. A method for testing a subject for a disease-related item, the method comprising:

a step of measuring the level or amount of follicular T cells reactive with a factor associated with the disease of the subject,

and comparing the level or amount with a predetermined reference.

2. The method of claim 1, wherein the benchmark is a benchmark with respect to at least 1 selected from the group consisting of: the history of the disease, the effectiveness of a prophylactic or vaccine against the disease, the effectiveness of a therapeutic against the disease, the ability to defend against a secondary disease, the condition of the disease, and the risk of developing the disease.

3. The method of claim 1 or 2, wherein the benchmark is a benchmark for the disease of the subject.

4. The method according to claim 1, wherein the criterion is a criterion for infection history of the disease of the subject.

5. The method of claim 1, wherein the benchmark is a benchmark for vaccine effectiveness, reinfection defense capacity of the disease of the subject.

6. The method of claim 1, wherein the benchmark is a benchmark for evaluating the effectiveness of a cancer immune drug against the disease of the subject.

7. The method according to claim 1, wherein the disease-related factor is an autoimmune disease-related factor, the determining comprising a step of determining the level or amount of follicular T cells reactive with the autoimmune disease-related factor, the criterion being a criterion for the risk of, or the condition of, an autoimmune disease associated with an autoantibody in the subject.

8. The method according to any one of claims 1 to 7, wherein the assay is performed according to enzyme-linked immunospot detection, flow cytometry, re-stimulation of peripheral blood mononuclear cells, PBMCs, new generation sequencer/genetic assay, combinatorial library analysis, immunostaining, pathological tissue examination.

9. The method of any of claims 1-8, wherein the comparing further comprises: comparing the measured level, spatial distribution or amount of follicular T cells reactive with the disease-related factor with the average or median level of the level, spatial distribution or amount of follicular T cells reactive with the disease-related factor of a subject not suffering from the disease.

10. The method of any one of claims 1-6 and 8-9, wherein the disease is an infection or cancer.

11. The method of any one of claims 1-6 and 8-9, wherein the disease is a viral infection.

12. The method of any one of claims 1-9, wherein the disease is an allergy or autoimmune disease.

13. A reagent or device for evaluating a disease in a subject, the reagent or device comprising a reagent or device for determining the level, spatial distribution or amount of follicular T cells reactive with a disease-related factor of the disease in the subject.

14. The reagent or device of claim 13, comprising a reagent that specifically reacts with follicular T cells.

15. The reagent or device according to claim 13, which is a device for performing enzyme-linked immunospot assays (ELISPOT), flow cytometry, re-stimulation assays of Peripheral Blood Mononuclear Cells (PBMCs), new generation sequencer/genetic tests (histology) or histology, immunostaining, in situ analysis.

16. The agent or device of any one of claims 13-15, wherein the disease is an infection or cancer.

17. The agent or device of any one of claims 13-15, wherein the disease is a viral infection.

18. The agent or device of any one of claims 13-15, wherein the disease is an allergy or autoimmune disease.

19. A system for evaluating a disease of a subject, the system comprising:

a reagent or device for determining the level, spatial distribution or amount of follicular T cells reactive with a disease-related factor of the disease in the subject,

and an evaluation unit for evaluating the disease based on the measurement result obtained by the reagent or the device.

20. The system of claim 19, wherein the evaluation portion is configured to perform at least 1 selected from the group consisting of: the evaluation of the history of the disease, the evaluation of the preventive or vaccine effectiveness of the disease, the evaluation of the effectiveness of the therapeutic agent of the disease, the evaluation of the ability to defend against the disease again, the evaluation of the condition of the disease, and the evaluation of the risk of developing the disease.

21. The system according to claim 19 or 20, further comprising an examination/diagnosis section for examining or diagnosing the disease of the subject.

22. The system according to claim 19, further comprising an infection history evaluation unit that evaluates an infection history of the disease of the subject.

23. The system according to claim 19, further comprising a vaccine effectiveness evaluation and reinfection defense capability evaluation unit for evaluating vaccine effectiveness and reinfection defense capability of the disease of the subject.

24. The system according to claim 19, further comprising a cancer immune drug effectiveness evaluation unit that evaluates effectiveness of a cancer immune drug of the disease of the subject.

25. The system according to claim 19, wherein the disease-related factor is an autoimmune disease-related factor, and the system comprises a follicular T cell measurement unit for measuring a level, spatial distribution, or amount of follicular T cells reactive with the autoimmune disease-related factor, and an autoimmune disease risk evaluation unit for evaluating an autoimmune disease associated with the autoantibody of the subject, and an autoimmune disease risk evaluation unit for evaluating a disease condition.

26. The system of any one of claims 19 to 25, wherein the reagent or device comprises a reagent that specifically reacts with follicular T cells.

27. The system according to any one of claims 19 to 25, wherein the device is a device for performing enzyme-linked immunospot assays (ELISPOT), flow cytometry, re-stimulation assays of Peripheral Blood Mononuclear Cells (PBMCs), new generation sequencer/genetic tests (histology) or immunostaining (pathology) or in situ analysis.

28. The system of any one of claims 19-27, the system further comprising: and a comparison unit for comparing the measured level, spatial distribution or amount of follicular T cells reactive with the disease-related factor with the average value or median of the levels, spatial distribution or amount of follicular T cells reactive with the disease-related factor of the subject not suffering from the disease.

29. The system of any one of claims 19-24 and 26-28, wherein the disease is an infection or cancer.

30. The system of any one of claims 19-24 and 26-28, wherein the disease is a viral infection.

31. The system of any one of claims 19-24 and 26-28, wherein the disease is an allergy or autoimmune disease.

32. A method for testing a subject for a disease-related item, the method comprising:

providing information on the level, spatial distribution or amount of the follicular T cells which are reactive with the disease-related factor of the disease in the subject,

33. The method of claim 32, wherein the benchmark is a benchmark with respect to at least 1 selected from the group consisting of: the history of the disease, the effectiveness of a prophylactic or vaccine against the disease, the effectiveness of a therapeutic against the disease, the ability to defend against the disease again, the disease assessment, and the risk of developing the disease.

34. The method according to claim 32, wherein the criterion is a criterion concerning the presence or absence of infection history of the disease in the subject.

35. The method of claim 32, wherein the benchmark is a benchmark for vaccine effectiveness, reinfection defense capacity of the subject for the disease.

36. The method of claim 32, wherein the benchmark is a benchmark for the effectiveness of a cancer immune drug of the disease of the subject.

37. The method of claim 32, wherein the disease-related factor is an autoimmune disease-related factor, the determining comprising the step of determining the level or amount of follicular T cells reactive with the autoimmune disease-related factor, the benchmark being with respect to the risk of, or the disease state of the subject for autoimmune diseases associated with autoantibodies.

38. The method according to any one of claims 32 to 37, wherein the step of providing information is performed according to enzyme-linked immunospot assay (ELISPOT), flow cytometry, re-stimulation of Peripheral Blood Mononuclear Cells (PBMCs), new generation sequencer/gene-assay (histologic assay), immunostaining (pathological tissue assay), in situ assay.

39. The method of any of claims 32-38, wherein the comparing further comprises: and comparing the information on the level, spatial distribution or amount of the follicular T cells reactive with the disease-related factor of the disease of the subject with the average value or median of the information on the level, spatial distribution or amount of the follicular T cells reactive with the disease-related factor of the disease of the subject not suffering from the disease.

40. The method of any one of claims 32-36 and 38-39, wherein the disease is an infection or cancer.

41. The method of any one of claims 32-36 and 38-39, wherein the disease is a viral infection.

42. The method of any one of claims 32-41, wherein the disease is an allergy or an autoimmune disease.

43. A program for installing a method for evaluating a disease of a subject in a computer, the program comprising computer-readable code, the program comprising:

providing information on the level, spatial distribution or amount of follicular T cells reactive with a disease-related factor of a disease in a subject,

and a step of evaluating information on the level, spatial distribution, or amount of the follicular T cells for the disease.

44. The program according to claim 43, wherein the evaluating step further comprises a step of performing at least 1 selected from the group consisting of: evaluation of history of the disease, evaluation of effectiveness of a prophylactic or vaccine for the disease, evaluation of effectiveness of a therapeutic for the disease, evaluation of defensive ability against a secondary disease, evaluation of a disease condition of the disease, and evaluation of risk of developing the disease.

45. The program according to claim 43, wherein the method comprises a step of evaluating infection histories of the disease in the subject.

46. The program according to claim 43, wherein the method comprises a step of evaluating vaccine effectiveness against the disease and a step of evaluating reinfection defense ability of the subject.

47. The program according to claim 43, wherein the method comprises the step of evaluating the effectiveness of a cancer immune drug against the disease in the subject.

48. The program of claim 43, wherein the disease-related factor is an autoimmune disease-related factor, the method comprising: the measurement is a step of measuring the level or amount of follicular T cells reactive with the autoimmune disease-related factor, and a step of evaluating the risk and disease condition of the autoimmune disease accompanied by the autoantibody in the subject.

49. The program according to any one of claims 43 to 48, wherein the step of providing information is performed according to enzyme-linked immunospot assay (ELISPOT), flow cytometry, re-stimulation of Peripheral Blood Mononuclear Cells (PBMC), new generation sequencer/gene-assay (ECP), immunostaining (pathological tissue assay), in situ assay.

50. The program of any one of claims 43 to 49, wherein the method further comprises: and comparing the information on the level, spatial distribution or amount of the follicular T cells reactive with the disease-related factor of the disease of the subject with the average value or median of the information on the level, spatial distribution or amount of the follicular T cells reactive with the disease-related factor of the disease of the subject not suffering from the disease.

51. The process of any one of claims 43-47 and 49-50, wherein the disease is an infection or cancer.

52. The process of any one of claims 43-47 and 49-50, wherein the disease is a viral infection.

53. The process of any one of claims 43 to 50, wherein the disease is an allergy or an autoimmune disease.

54. A computer-readable recording medium having stored therein the program of any one of claims 43 to 53.

55. A method for evaluating a disease in a subject, the method comprising the step of determining the level or amount of follicular T cells in the subject that are reactive with a disease-related factor of the disease.

56. A method for evaluating a disease in a subject, the method comprising the steps of providing information about the level, spatial distribution or amount of follicular T cells reactive with a disease-related factor of the disease in the subject, and evaluating the information about the level, spatial distribution or amount of follicular T cells for the disease.